U.S. patent application number 11/768103 was filed with the patent office on 2012-05-17 for pathogen detection by simultaneous size/fluorescence measurement.
Invention is credited to Jian-Ping Jiang.
Application Number | 20120120385 11/768103 |
Document ID | / |
Family ID | 39721740 |
Filed Date | 2012-05-17 |
United States Patent
Application |
20120120385 |
Kind Code |
A1 |
Jiang; Jian-Ping |
May 17, 2012 |
PATHOGEN DETECTION BY SIMULTANEOUS SIZE/FLUORESCENCE
MEASUREMENT
Abstract
A method and apparatus for detecting pathogens and particles in
a fluid in which particle size and intrinsic fluorescence of a
simple particle is determined.
Inventors: |
Jiang; Jian-Ping; (Tucson,
AZ) |
Family ID: |
39721740 |
Appl. No.: |
11/768103 |
Filed: |
June 25, 2007 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60805962 |
Jun 27, 2006 |
|
|
|
Current U.S.
Class: |
356/51 ;
356/72 |
Current CPC
Class: |
G01N 15/1456 20130101;
G01N 2021/4707 20130101; G01N 21/64 20130101; G01N 2015/0088
20130101; C12Q 1/04 20130101; G01N 21/51 20130101; G01N 2015/1493
20130101 |
Class at
Publication: |
356/51 ;
356/72 |
International
Class: |
G01N 21/00 20060101
G01N021/00 |
Claims
1. A method of differentiating biological particles from inert
particles in a fluid which comprises simultaneously measuring a
particle size and detecting intrinsic fluorescence from that
particle.
2. The method of claim 1, wherein fluorescence intensity is
measured and assigned a value, and including the step of
classifying the particle as either inert or biological based on
particle size and fluorescence intensity.
3. The method of claim 2, wherein size information of the particle
is used to classify whether that particle is a microorganism.
4. The method of claim 3, wherein size information of the particle
is derived from determining cross-section area of the particle, or
volume of the particle.
5. The method of claim 4, wherein volume of the particle is derived
by first determining the diameter of the particle, and calculating
its volume based on said diameter.
6. The method of claim 2, wherein particle size and fluorescence
intensity data from an individual particle is used to differentiate
between pollen and allergens from microbes.
7. The method of claim 2, wherein particle size and fluorescence
signal data from an individual particle is used to estimate
relative abundance of biochemical compounds inside the biological
particles.
8. The method of claim 2, wherein particle size and fluorescence
intensity value from an individual particle is normalized by its
size or volume and used to differentiate between inert particles
from microbes.
9. The method of claim 2, wherein particle size and fluorescence
intensity value from an individual particle is normalized by its
size or volume and used to differentiate between pollen and
allergens from microbes.
10. The method of claim 1, wherein the fluid comprises air.
11. The method of claim 1, wherein the fluid comprises water.
12. A method for detecting and classifying a particle in a liquid
or gas comprising illuminating the particle with a UV light source,
and simultaneously measuring a size of the particle and any
intrinsic fluorescence from the particle.
13. The method of claim 12, wherein the particle comprises a
bioparticle.
14. The method of claim 13, wherein the bioparticle comprises a
microbe.
15. The method of claim 12, wherein the bioparticle is selected
from the group consisting of a bacterium, a mold, a fungi, and a
spore.
16. The method of claim 12, including the step of measuring
fluorescence intensity.
17. The method of claim 12, including the step of comparing
particle size information and fluorescence intensity to classify
the particle as inert or microbial in origin.
18. The method of claim 12, including the step of differentiating
the particle as a bacterium, a mold, a fungi or a spore.
19. The method of claim 12, including the step of differentiating
the particle as a pollen or an allergen.
20. The method of claim 18, including the step of classifying the
particle based on its fluorescence response.
21. The method of claim 19, including the steps of classifying the
particle based on its fluorescence response.
22. The method of claim 18, including the steps of classifying the
particle based on its diameter or volume.
23. The method of claim 18, including the steps of classifying the
particle based on its fluorescence intensity normalized by its
diameter or volume.
24. The method of claim 19, including the steps of classifying the
particle based on its diameter or volume.
25. The method of claim 19, including the steps of classifying the
particle based on its fluorescence intensity normalized by its
diameter or volume.
26. A particle detector system, comprising: a sample cell; a light
source on one side of a sample cell for sending a focused beam of
light through the sample, whereby portions of the beam of light are
scattered at various angles by particles of various sizes present
in the sample area, and an unscattered portion of the beam of light
remains unscattered; a beam blocking device on an opposite side of
the sample cell for blocking at least the portion of the
unscattered portion of the beam of light and for limiting a range
of particles measured; a first detector positioned in the light
path after the beam blocking device for detecting a portion of
forward scattered light, and producing an output including
information on the size of a single particle in the light path
within a predetermined size range; a second detector positioned off
axis from the beam of light for detecting intrinsic fluorescence
from said same single particle.
27. The system of claim 26, wherein an elliptical mirror is located
in a particle sampling region such that an intersection of the
incoming particle stream and the light beam are at one foci of the
ellipsoid, and the second detector is at the other foci.
28. The system of claim 26, further comprising an alarm unit for
providing a warning signal when a particle within a predetermined
size range is detected which also fluoresces.
29. The system of claim 26, wherein the light source emits
ultraviolet radiation.
30. The system of claim 26, wherein the light source comprises a
LED.
31. The system of claim 30, further comprising a collimator lens
optically positioned between the light source and the first
detector.
32. The system of claim 26, further comprising a processing unit
for processing particle size distribution and particle fluorescence
at a give time, and displaying a histogram of the particle on an
output device.
33. The system of claim 26, wherein the first detector comprise a
photodiode.
34. The system of claim 26, wherein the sample cell comprises an
air sample cell.
35. The system of claim 26, wherein the sample cell comprises a
water sample cell.
36. The system of claim 26, further comprising computer readable
program code for integrating detected particle size and detected
intrinsic fluorescence.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims priority from U.S. Provisional
Application Ser. No. 60/805,962 filed Jun. 27, 2006.
FIELD OF THE INVENTION
[0002] The present invention relates generally to a system and
method for detecting airborne or waterborne particles, and more
particularly to a system and method for detecting airborne or
waterborne particles and classifying the detected particles. The
invention has particular utility in detecting and classifying
allergens and biological warfare agents and will be described in
connection with such utility, although other utilities are
contemplated.
BACKGROUND OF THE INVENTION
[0003] An urban terrorist attack involving release of biological
warfare agents such as bacillus anthracis (anthrax) is presently a
realistic concern. Weaponized anthrax spores are extremely
dangerous because they can gain passage into the human lungs. A
lethal inhalation dose of anthrax spores for humans, LD.sub.50
(lethal dose sufficient to kill 50% of the persons exposed) is
estimated to be 2,500 to 50,000 spores (see T. V. Inglesby, et al.,
"Anthrax as a Biological Weapon", JAMA, vol. 281, page 1735, 1999).
Some other potential weaponized bio-agents are yersinia pestis
(plague), clostridium botulinum (botulism), and francisella
tularensis. In view of this potential threat, there is currently a
need for an early warning system to detect such an attack. In the
pharmaceutical, healthcare and food industries, a real time
detector of environmental microbial level is useful for public
health, quality control and regulatory purposes. For example,
parenteral drug manufacturers are required to monitor the microbial
levels in their aseptic clean rooms. In these applications, an
instrument which can detect microbes in the environment
instantaneously will be a useful tool and have advantages over
conventional nutrient plate culture methods which requires days for
microbes to grow and to be detected.
[0004] Particle size measurement and ultraviolet (UV) induced
fluorescence detection have been used to detect the presence of
biological substances in the air. There exist various patents
describing using these techniques as early warning sensors for
bio-terrorist attack release of weaponized bio-agents. Among these
devices are Biological Agent Warning Sensor (BAWS) developed by MIT
Lincoln Laboratory, fluorescence biological particle detection
system of Ho (Jim yew-Wah Ho, U.S. Pat. Nos. 5,701,012; 5,895,922;
6,831,279); FLAPS and UV-APS by TSI of Minnesota (Peter P.
Hairston; and Frederick R. Quant; U.S. Pat. No. 5,999,250), and a
fluorescence sensor by Silcott (U.S. Pat. No. 6,885,440).
[0005] A proposed bio-sensor based on laser-induced fluorescence
using a pulsed UV laser is described by T. H. Jeys, et al., Proc.
IRIS Active Systems, vol. 1, p. 235, 1998. This is capable of
detecting an aerosol concentration of five particles per liter of
air, but involves expensive and delicate instruments. Other
particle counters are manufactured by Met One Instrument, Inc, of
Grants Pass, Oreg., Particle Measurement Systems, Inc., of Boulder,
Colo., and Terra Universal Corp., of Anaheim, Calif.
[0006] Various detectors have been designed to detect airborne
allergen particles and provide warning to sensitive individuals
when the number of particles within an air sample exceeds a
predetermined minimum value. These are described in U.S. Pat. Nos.
5,646,597, 5,969,622, 5,986,555, 6,008,729, 6,087,947, and
7,053,783, all to Hamburger et al. These detectors all involve
direction of a light beam through a sample of environmental air
such that part of the beam will be scattered by any particles in
the air, a beam blocking device for transmitting only light
scattered in a predetermined angular range corresponding to the
predetermined allergen size range, and a detector for detecting the
transmitted light.
SUMMARY OF THE INVENTION
[0007] For the purpose of detection of microbes in air or water, it
is of importance to devise an effective system to measure both
particle size and fluorescence generated intrinsically by the
microbes. The present invention provides a sensor system which is
capable of simultaneously measuring particle size and detecting the
presence of intrinsic fluorescence from metabolites and other
bio-molecules, on a particle-by-particle basis. The advantages of
this detection scheme over the prior art are several. For one it
provides a deterministic particle measurement methodology for
characterizing particles rather than relying on statistical models
employed in prior art for particle characterization. The
deterministic measurement methodology enables more definitive
assignment of particle characters than the prior art and less
reliance on statistical models. It also reduces the possibility of
false positives in microbial detection, for example, pollen (larger
sizes than microbes) and smoke particles (smaller sizes than
microbes) can be excluded from detection. And, it allows detailed
analyses of data collected on each individual particle for
characterizing the particle, such as intensity of fluorescence
signal from a particle as a function of its cross-sectional area or
volume, for the purpose of determining the biological status of the
particles.
[0008] The current invention comprises three main components: (1) a
first optical system for measuring an individual particle size; (2)
a second optical system to detect a UV laser-induced intrinsic
fluorescence signal from an individual particle; and (3) a data
recording format for assigning both particle size and fluorescence
intensity to an individual particle, and computer readable program
code for differentiating microbes from non-microbes (e.g. inert
dust particles).
[0009] The optical assembly of the present invention has two
optical sub-assemblies: (a) an optical setup to measure the
particle size. As an example, the preferred embodiment of the
current invention uses the well-known and often used Mie scattering
detection scheme, but applies it in a novel way, enabling the
system to make highly accurate measurements of airborne particles
with size ranges from 0.5 microns to 20 microns. This capability to
make fine distinctions in size is important in order to determine
the class of microbe, because different classes of microbes have
different size ranges; (b) simultaneous to the particle size
measurement, an optical apparatus is used to measure the
fluorescence level from the particle being interrogated. As an
example, the preferred embodiment of the current invention uses an
elliptical mirror which is positioned to collected fluorescence
emission from the same particle as it is being measured for
size.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] Further features and advantages of the present invention
will be seen from the following detailed description, taken in
conjunction with the accompanying drawings, wherein:
[0011] FIG. 1 is a plot showing particle size ranges of several
airborne inert and microbial particulates;
[0012] FIG. 2(a) is a histogram representation of simultaneous
measurements of particle size and fluorescence showing particle
distribution for microbe-free air;
[0013] FIG. 2(b) is a histogram showing simultaneous measurements
of particle size and fluorescence for air containing Baker's yeast
powder;
[0014] FIG. 3 is a histogram representation of simultaneous
measurements of 7 micron size fluorescent dye doped particles and
fluorescence;
[0015] FIG. 4 is a schematic diagram of an optical system in
accordance with the present invention, for performing simultaneous
measurements of particle size and fluorescence; and
[0016] FIG. 5 is a block diagram of the optical system of FIG.
4.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0017] FIG. 4 is a schematic representation of an optical system
for a fluid particle detector system according to a first exemplary
embodiment of the invention. This first exemplary embodiment of the
system is designed, for example to detect airborne or waterborne
bio-terrorist agents deliberately released by terrorists or others,
but also may be used in civilian applications to detect harmful
levels of other airborne or waterborne particles which may exist
naturally such as mold or bacteria, or which may have been
accidentally, inadvertently, naturally, or deliberately related, or
for other industrial applications such as the food and
pharmaceutical manufacturing industries, as well as clean room
applications.
[0018] The term "fluid borne particles" as used herein means both
airborne particles and waterborne particles.
[0019] The term "pathogen" as used herein refers to any airborne or
waterborne particles, biological agent, or toxin, which could
potentially harm or even kill humans exposed to such particles if
present in the air or water in sufficient quantities.
[0020] The term "biological agent" is defined as any microorganism,
pathogen, or infectious substance, toxin, biological toxin, or any
naturally occurring, bioengineered or synthesized component of any
such micro-organism, pathogen, or infectious substance, whatever
its origin or method of production. Such biological agents include,
for example, biological toxins, bacteria, viruses, rickettsiae,
spores, fungi, and protozoa, as well as others known in the
art.
[0021] "Biological toxins" are poisonous substances produced or
derived from living plants, animals or microorganisms, but also can
be produced or altered by chemical means. A toxin, however,
generally develops naturally in a host organism (i.e., saxitoxin is
produced by marine algae), but genetically altered and/or
synthetically manufactured toxins have been produced in a
laboratory environment. Compared with microorganisms, toxins have a
relatively simple biochemical composition and are not able to
reproduce themselves. In many aspects, they are comparable to
chemical agents. Such biological toxins are, for example, botulinum
and tetanus toxins, staphylococcal enterotoxin B, tricothocene
mycotoxins, ricin, saxitoxin, Shiga and Shiga-like toxins,
dendrotoxins, erabutoxin b, as well as other known toxins.
[0022] The detector system of the present invention is designed to
detect airborne or waterborne particles and produce outputs
indicating, for instance, the number of particles of each size
within the range, which is detected in a sample, and indicate
whether the particles are biologic or non-biologic. The system also
may produce an alarm signal or other response if the number of
particles exceeds a predetermined value above a normal background
level, and/or biological organisms or biological agents and
potentially dangerous.
[0023] FIG. 4 is a representation of system 10 for a fluid particle
detector system according to an exemplary embodiment of the
invention. As shown in FIG. 4, the system 10 includes an UV light
excitation source 12 such as a laser providing a beam of
electromagnetic radiation 14 have an UV light source wavelength.
The UV light source is selected to have a wavelength capable of
exciting intrinsic fluorescence from metabolites inside microbes.
By way of example, the excitation source 12 preferably operates in
a wavelength of about 270 nm to about 410 nm, preferably about 350
nm to about 410 nm. A wavelength of about 270 nm to about 410 nm is
chosen based on the premise that microbes comprise three primary
metabolites: tryptophan, which normally fluoresces at about 270 nm
with a range of about 220 nm-about 300 nm; nicotinamide adenine
dinucleotide (NADH) which normally fluoresces at about 340 nm
(range about 320 nm-about 420 nm); and riboflavin which normally
fluoresces at about 400 nm (range about 320 nm-about 420 nm).
Preferably, however, the excitation source 12 has a wavelength of
about 350 to about 410 nm. This wavelength ensures excitation of
two of the three aforesaid primary metabolites, NADH, and
riboflavin in bio-agents, but excludes excitation of interferences
such as from diesel engine exhaust and other inert particles such
as dust or baby powder. Thus, in a preferred embodiment the present
invention makes a judicial selection of wavelength range of the
excitation source 12, which retains the ability of exciting
fluorescence from NADH and riboflavin (foregoing the ability to
excite tryptophan) while excluding the excitation of interferents
such as diesel engine exhaust. This step is taken to reduce false
alarms generated by diesel exhaust (which can be excited by short
UV wavelengths such as 266 nm light.
[0024] In the system 10 illustrated in FIG. 4, environmental air
(or a liquid sample) is drawn into the system through a nozzle 16
for particle sampling. Nozzle 16 has an opening 18 in its middle
section to allow the laser beam to pass through the particle
stream. Directly downstream from the laser beam is a Mie scattering
particle-size detector 20. Mie scattering particle-size detector 20
includes a beam blocker lens 22, a collimator lens 24 and a
condenser lens 26 for focusing a portion of the light beam 14 onto
a particle detector 28.
[0025] Off axis from the laser beam 14, an elliptical mirror 30 is
placed at the particle-sampling region in such a way that the
intersection of the incoming particle stream and the laser beam is
at one of the two foci of the ellipsoid, while a fluorescence
detector 32 (in this case a photo-multiplier tube) occupies the
other focus. This design utilizes the fact that a point source of
light emanating from one of the two foci of an ellipsoid will be
focused onto the other. In this optical design, the elliptical
mirror 30 concentrates the fluorescence signal from microbe and
focus it onto the fluorescence detector 32. An optical filter 34 is
placed in front of the fluorescence detector to block the scattered
UV light and pass the induced fluorescence.
[0026] The beam blocker lens 22 is designed to reflect
non-scattered elements of the laser beam 14, and may have a
material, such as vinyl, attached a front surface to reflect the
non-scattered elements of the beam of electromagnetic radiation.
Other features and considerations for the beam blocker lens 22 are
disclosed in some of the earlier U.S. patents to Hamburger et al.
listed above, and in PCT Application Serial No. PCT/US2006027638,
incorporated herein by reference.
[0027] The particle detector 20 may comprise, for example, a
photodiode for sizing the particles, e.g. as described in the
earlier U.S. patent to Hamburger et al., listed above, and
incorporated herein by reference.
[0028] The present invention's use of Mie scattering also
facilitates the placement of optical components for the detection
of UV light illumination to concurrently examine individual
particles for the presence of the metabolites NADH, riboflavin and
other bio-molecules, which are necessary intermediates for
metabolism of living organisms, and therefore exist in microbes
such as bacteria and fungi. If these chemical compounds exist in a
bio-aerosol, they are excited by the UV photon energy and
subsequently emit auto-fluorescence light which may be detected by
an instrument based on the detection scheme outlined above. While
this detection scheme is not capable of identifying the genus or
species of microbes, and viruses may be too small and lack the
metabolism for detection, this detection scheme's ability to
simultaneously and for each particle determine the size of the
particle and if it is biologic or inert indicates to the user the
presence or absence of microbial contamination.
[0029] Referring to FIG. 5, the functionality of the simultaneous
particle sizing and fluorescence measurement scheme of the present
invention is depicted in the graphic presentation of the
measurement results from such as an instrument. The principle of
operation is as follows: an instrument continuously monitors the
environmental air (or liquid) to measure the size of each
individual airborne particle in real time and to concurrently
determine whether that particle emits fluorescence or not. A
threshold is set for the fluorescence signal. If the fluorescence
signal is below the set level, the particle is marked inert. This
fluorescence signal threshold can be fluorescence signal intensity,
fluorescence intensity as a function of particle cross-sectional
area or a function of particle volume. If the fluorescence signal
threshold exceeds the set level, the particle is marked biological.
The combined data of particle size and fluorescence signal strength
will determine the presence or absence of microbes on a
particle-by-particle basis. FIGS. 2(a) and 2(b) illustrate the
functionality of a detector in accordance with the present
invention. They show the environmental airborne particle data
measured by using this detection scheme. In each graph, the upper
part depicts in logarithmic scale the particle size histogram of
particle concentration (#/liter of air) versus particle size (from
1 micron to 13 microns); solid bars represent inert particles
whereas striped bars indicate the presence of microbes. The lower
part of the graph is a real-time snap shot of the particles
detected within 1 second: each spike represents one single particle
and its height corresponds to the particle size. In FIG. 2(a), the
test was done for clean air, so there were only inert particles,
free from microbes. In a second test, Baker's yeast powder
(Saccharomyces cerevisiae) was released into the air. The presence
of the microbe was detected and shown by the striped bars in the
histogram in FIG. 2(b).
[0030] FIG. 3 shows the data set obtained when 7 microns
fluorescent dye doped plastic beads were disseminated into a
detector capable of simultaneous particle size and fluorescence
measurement scheme. The striped bars show the presence of
fluorescence in those particles with a distribution in the 7
microns size range.
[0031] It should be emphasized that the above-described embodiments
of the present invention, particularly, any "preferred"
embodiments, are merely possible examples of implementations,
merely set forth for a clear understanding of the principles of the
invention. Many variations and modifications may be made to the
above-described embodiments of the invention without departing
substantially from the spirit and principles of the invention. All
such modifications and variations are intended to be included
herein within the scope of this disclosure and the present
invention and protected by the following claims.
* * * * *