U.S. patent application number 13/211940 was filed with the patent office on 2012-02-23 for fiber sampler for recovery of bioaerosols and particles.
This patent application is currently assigned to Reseach Triangle Institute. Invention is credited to David S. ENSOR, Karin K. Foarde, Howard J. Walls.
Application Number | 20120045752 13/211940 |
Document ID | / |
Family ID | 45594361 |
Filed Date | 2012-02-23 |
United States Patent
Application |
20120045752 |
Kind Code |
A1 |
ENSOR; David S. ; et
al. |
February 23, 2012 |
FIBER SAMPLER FOR RECOVERY OF BIOAEROSOLS AND PARTICLES
Abstract
A bioparticle collection device and an aerosol collection
system. The bioparticle collection device includes a collection
medium including a plurality of fibers formed into a fiber mat and
configured to collect bioparticles thereon, and includes a
viability enhancing material provider disposed in a vicinity of the
plurality of fibers and configured to provide a viability enhancing
material to the collected bioparticles to maintain viability of the
bioparticles collected by the fiber mat. The aerosol collection
system includes an aerosol pumping device configured to entrain
particles in an gas stream, an aerosol saturation device configured
to saturate the particles in the gas stream with a biocompatible
liquid, and an aerosol collection medium downstream from the
aerosol saturation device and including a plurality of fibers
formed into a fiber mat for collection of the saturated aerosol
particles.
Inventors: |
ENSOR; David S.; (Chapel
Hill, NC) ; Walls; Howard J.; (Apex, NC) ;
Foarde; Karin K.; (Chapel Hill, NC) |
Assignee: |
Reseach Triangle Institute
Research Triangle Park
NC
|
Family ID: |
45594361 |
Appl. No.: |
13/211940 |
Filed: |
August 17, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61374466 |
Aug 17, 2010 |
|
|
|
Current U.S.
Class: |
435/5 ;
435/297.1; 435/34; 977/788; 977/903 |
Current CPC
Class: |
G01N 2001/2223 20130101;
B82Y 15/00 20130101; G01N 1/2205 20130101; G01N 1/2208 20130101;
G01N 1/2214 20130101; G01N 33/497 20130101; B01D 2239/025 20130101;
B01D 2239/0604 20130101; C12Q 1/24 20130101 |
Class at
Publication: |
435/5 ;
435/297.1; 435/34; 977/788; 977/903 |
International
Class: |
C12Q 1/70 20060101
C12Q001/70; C12Q 1/04 20060101 C12Q001/04; C12M 1/12 20060101
C12M001/12 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0003] This invention was made with government support under
HSHQDC-09-C-00154 awarded by DHS. The government has certain rights
in the invention
Claims
1. A bioparticle collection device comprising: a collection medium
including a plurality of fibers formed into a fiber mat and
configured to collect bioparticles thereon; and a viability
enhancing material provider disposed in a vicinity of the plurality
of fibers and configured to provide a viability enhancing material
to the collected bioparticles to maintain viability of the
bioparticles collected by the fiber mat.
2. The device of claim 1, wherein the viability enhancing material
provider comprises an osmotic material disposed in contact with the
plurality of fibers and configured to maintain a relative humidity
suitable for said viability of bioparticles.
3. The device of claim 2, wherein the osmotic material comprises a
water-regulating material configured to provide water to the
fibers.
4. The device of claim 1, wherein the osmotic material comprises a
nutrient supply providing nutrients to support biological viability
of biomaterial collected in the fiber mat.
5. The device of claim 4, wherein the nutrient supply comprises a
supply of at least one of water, proteins, carbohydrates, sugars,
salts, phosphate buffered saline, and tryptic soy broth.
6. The device of claim 1, wherein the collection medium and the
viability enhancing material are disposed in one of an air filter,
a wipe, a brush, a swab, a sorbent pad, or a liquid filter.
7. The device of claim 1, wherein the plurality of fibers comprise
materials dissolvable in a bio-compatible solvent.
8. The device of claim 1, further comprising a support supporting
the collection medium.
9. The device of claim 8, wherein the support comprises: at least
one of a filter, a plastic foam, a metallic foam, a semi-conductive
foam, a woven material, a nonwoven material, a plastic screen, a
textile, and a high efficiency particulate air (HEPA) filter
medium.
10. The device of claim 1, wherein the viability enhancing material
provider comprises at least a part of the plurality of fibers.
11. The device of claim 1, wherein the viability enhancing material
provider comprises a material different from the plurality of
fibers.
12. The device of claim 1, wherein the fibers have an average fiber
diameter of less than 10 microns.
13. The device of claim 1, wherein the fiber mat comprises between
4 to 4000 layers of the fibers.
14. The device of claim 1, wherein the fiber mat comprises a
thickness between 0.25 and 500 .mu.m.
15. The device of claim 1, wherein the fibers comprise a pH
responsive material.
16. The device of claim 1, wherein the fibers comprise at least one
of acrylonitrile/butadiene copolymer, cellulose, cellulose acetate,
chitosan, collagen, DNA, fibrinogen, fibronectin, nylon,
poly(acrylic acid), poly(chloro styrene), poly(dimethyl siloxane),
poly(ether imide), poly(ether sulfone), poly(ethyl acrylate),
poly(ethyl vinyl acetate), poly(ethyl-co-vinyl acetate),
poly(ethylene oxide), poly(ethylene terephthalate), poly(lactic
acid-co-glycolic acid), poly(methacrylic acid) salt, poly(methyl
methacrylate), poly(methyl styrene), poly(styrene sulfonic acid)
salt, poly(styrene sulfonyl fluoride),
poly(styrene-co-acrylonitrile), poly(styrene-co-butadiene),
poly(styrene-co-divinyl benzene), poly(vinyl acetate), poly(vinyl
alcohol), poly(vinyl chloride), poly(vinylidene fluoride),
polyacrylamide, polyacrylonitrile, polyamide, polyaniline,
polybenzimidazole, polycaprolactone, polycarbonate, polyurethanes,
poly(dimethylsiloxane-co-polyethyleneoxide),
poly(etheretherketone), polyethylene, polyethyleneimine, polyimide,
polyisoprene, polylactide, polypropylene, polystyrene, polysulfone,
polyurethane, poly(vinylpyrrolidone), poly(2-hydroxy ethyl
methacrylate) (PHEMA), gelatin, proteins, SEBS copolymer, silk
(natural or synthetically derived), styrene/isoprene copolymer, and
polymer blends including at least one of poly(vinylidene
fluoride)-blend-poly(methyl methacrylate),
polystyrene-blend-poly(vinylmethylether), poly(methyl
methacrylate)-blend-poly(ethyleneoxide), poly(hydroxypropyl
methacrylate)-blend poly(vinylpyrrolidone),
poly(hydroxybutyrate)-blend-poly(ethylene oxide), protein
blend-polyethyleneoxide, polylactide-blend-polyvinylpyrrolidone,
polystyrene-blend-polyester, polyester-blend-poly(hydroxyethyl
methacrylate), poly(ethylene oxide)-blend poly(methyl
methacrylate), and poly(hydroxystyrene)-blend-poly(ethylene
oxide)).
17. The device of claim 1, wherein the fibers comprise at least one
of polyethylene oxide (PEO), poly(2-hydroxy ethyl methacrylate)
(PHEMA), gelatine, and poly(vinyl alcohol), cellulose acetate,
chitosan, poly(lactic acid), collagen, poly caprolactone,
poly(ethylene glycol) methyl ether, polystyrene.
18. The device of claim 1, wherein the viability enhancing material
provider is intermixed with the plurality of fibers.
19. The device of claim 1, wherein the viability enhancing material
comprises a layer of biomaterial separated from the plurality of
fibers.
20. The device of claim 1, wherein the viability enhancing material
provider comprises at least one of a polycarbonate, Eudragit
polymers, copolymers of N-isopropyl acrylamide (NIPAM) and
N-methyacryloy-L-Leucine (MALEU) or (N,N-dimethylamino)ethyl
methacrylate (DMAEMA), cellulose, and degraded cellulose.
21. An aerosol collection system comprising: an aerosol pumping
device configured to entrain particles in an gas stream; an aerosol
saturation device configured to saturate the particles in the gas
stream with a biocompatible liquid; and an aerosol collection
medium downstream from the aerosol saturation device and including,
a plurality of fibers formed into a fiber mat for collection of the
saturated aerosol particles, and a viability enhancing material
provider disposed in a vicinity of the plurality of fibers and
configured to provide a viability enhancing material to the
collected bioparticles to maintain viability of the bioparticles
collected by the fiber mat.
22. The system of claim 21, further comprising a humidity control
device configured to maintain the collection medium at a relative
humidity from 50 to 100%.
23. The system of claim 21, further comprising a humidity control
device configured to maintain the collection medium at a relative
humidity from 65 to 85%.
24. The system of claim 21, further comprising a humidity control
device configured to maintain the collection medium at a relative
humidity from 75 to 81%.
25. The system of claim 21, wherein the aerosol collection medium
comprise at least one of a flow-through or an impaction device.
26. The system of claim 21, wherein the viability enhancing
material provider comprises an osmotic material disposed in contact
with the plurality of fibers and configured to maintain a relative
humidity suitable for said viability of bioparticles.
27. The system of claim 26, wherein the osmotic material comprises
a water-regulating material configured to provide water to the
fibers.
28. The system of claim 26, wherein the osmotic material comprises
a nutrient supply providing nutrients to support biological
viability of biomaterial collected in the filtration medium.
29. The system of claim 28, wherein the nutrient supply comprises a
supply of at least one of proteins, sugars, and salts.
30. The system of claim 21, wherein the plurality of fibers
comprise nanofibers.
31. The system of claim 21, wherein the plurality of fibers
comprise materials dissolvable in a bio-compatible solvent.
32. The system of claim 31, further comprising a support supporting
the collection medium.
33. The system of claim 32, wherein the support comprises: at least
one of a filter, a plastic foam, a metallic foam, a semi-conductive
foam, a woven material, a nonwoven material, a plastic screen, a
textile, and a high efficiency particulate air (HEPA) filter
medium.
34. The system of claim 21, wherein the viability enhancing
material provider comprises at least a part of the plurality of
fibers.
35. The system of claim 21, wherein the viability enhancing
material provider comprises a material different from the plurality
of fibers.
36. The system of claim 21, wherein the fibers have an average
fiber diameter of less than 10 microns.
37. The system of claim 21, wherein the fiber mat comprises a
thickness between 0.25 and 500 .mu.m.
38. The system of claim 21, wherein the fibers comprise a pH
sensitive material.
39. The system of claim 21, wherein the viability enhancing
material provider is intermixed with the plurality of fibers.
40. The system of claim 21, wherein the viability enhancing
material comprises a separate layer from a layer including the
plurality of fibers.
41. The system of claim 21, wherein the aerosol collection medium
comprises at least one of a flow-through medium or an impaction
medium.
42. A method for collecting aerosols, comprising: entraining
particles in an gas stream; saturating the particles in the gas
stream with a biocompatible liquid; and collecting the saturated
aerosol particles by a collection medium including a plurality of
fibers formed into a fiber mat including and a viability enhancing
material provider disposed in a vicinity of the plurality of fibers
and configured to provide a viability enhancing material to the
collected bioparticles to maintain viability of the bioparticles
collected by the fiber mat.
43. The method of claim 42, further comprising injecting the
viability enhancing material into the collection medium prior to
collecting the aerosol particles.
44. The method of claim 43, wherein the injecting comprises
injecting at least one of water, proteins, carbohydrates, sugars,
salts, phosphate buffered saline, and tryptic soy broth.
45. The method of claim 42, further comprising injecting the
viability enhancing material into the collection medium during the
collecting of the aerosol particles.
46. The method of claim 45, wherein the injecting comprises
injecting at least one of water, proteins, carbohydrates, sugars,
salts, phosphate buffered saline, and tryptic soy broth.
47. The method of claim 42, further comprising introducing
antioxidant gases into the collection medium.
48. A bioparticle collection device comprising: a collection medium
including a plurality of fibers formed into a fiber mat; and an
osmotic material disposed in contact with the plurality of
fibers.
49. An aerosol collection system comprising: an aerosol pumping
device configured to entrain particles in an gas stream; an aerosol
saturation device configured to saturate the particles in the gas
stream with a biocompatible liquid; and an aerosol collection
medium downstream from the aerosol saturation device and including,
a plurality of fibers formed into a fiber mat for collection of the
saturated aerosol particles, and an osmotic material disposed in
contact with the plurality of fibers.
50. A method for collecting aerosols, comprising: entraining
particles in an gas stream; saturating the particles in the gas
stream with a biocompatible liquid; and collecting the saturated
aerosol particles by a collection medium including a plurality of
fibers formed into a fiber mat including and an osmotic material
disposed in contact with the plurality of fibers.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is related to and claims priority under 35
U.S.C. 119(e) to U.S. Application Ser. No. 61/374,466, filed Aug.
17, 2010, entitled "Fiber Sampler for Recovery of Bioaerosols and
Particles," the entire contents of which are incorporated herein by
reference.
[0002] This application is related to U.S. application Ser. No.
11/559,282, filed on Nov. 13, 2006, entitled "Particle Filter
System Incorporating Nanofibers," the entire contents of which are
incorporated herein by reference. This application is related to
U.S. application Ser. No. 10/819,916, filed on Apr. 8, 2004,
entitled "Electrospinning of Polymer Nanofibers Using a Rotating
Spray Head," the entire contents of which are incorporated herein
by reference. This application is also related to U.S. application
Ser. No. 10/819,942, filed on Apr. 8, 2004, entitled
"Electrospray/electrospinning Apparatus and Method," the entire
contents of which are incorporated herein by reference. This
application is related to U.S. application Ser. No. 10/819,945,
filed Apr. 8, 2004, entitled "Electrospinning in a Controlled
Gaseous Environment," the entire contents of which are incorporated
herein by reference. This application is related to U.S. Ser. No.
11/130,269, filed May 17, 2005 entitled "Nanofiber Mats and
Production Methods Thereof," the entire contents of which are
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0004] 1. Field of the Invention
[0005] The invention is related to fibers, methods, and devices for
collection of bioaerosols and particles on fiber structures. The
invention is also related to electrospun materials for filtration
and air sampling, in particular the collection of bioaerosols.
[0006] 2. Description of the Related Art
[0007] Collection of both indoor and outdoor air samples is
important for monitoring air quality. A wide range of
microorganisms are of interest including bacteria, fungi and
viruses. From a health standpoint, toxins and allergens may be of
interest as well. For example see, J. M. Macher (1999) Bioaerosols,
Assessment and Control, American conference of Governmental
Industrial Hygienists, Cincinnati, Ohio.
[0008] More recently, concerns about airborne pathogens being
present due to natural processes, accidents, or terrorist attacks
has led to the need for improved sampling systems. In addition to
the problem of collecting the aerosol (particles) is the problem of
recovering the particles for analysis. In the case of biological
particles, a common problem is that the organisms die during
collection or after collection while awaiting laboratory analysis.
Current sampling methods onto microbiological media do not permit
extended sampling times beyond 30-45 minutes in the case where
preservation of viable organisms is of interest.
[0009] In general, a concentrated, viable collect of submicrometer
biological particles has been recognized in the art as a challenge.
Each bioaerosol sampling method has limitations with respect to
sampling time, desiccation, shelf life of sample, complexity,
compatibility with analysis via PCR and live recovery. Some
evaluations are given by Griffiths and Decosemo (1994); Henningson
and Ahlberg (1994); Wang, Reponen et al. (2001); Tseng and Li
(2005); Verreault, Moineau et al. (2008); Mainelis and Tabayoyong
(2010) listed below: [0010] Griffiths, W. D. and G. A. L. Decosemo
1994. The Assessment of Bioaerosols--a Critical-Review. Journal of
Aerosol Science 25(8): 1425-1458. [0011] Henningson, E. W. and M.
S. Ahlberg 1994. Evaluation of Microbiological Aerosol Samplers--a
Review. Journal of Aerosol Science 25(8): 1459-1492. [0012]
Mainelis, G. and M. Tabayoyong 2010. The Effect of Sampling Time on
the Overall Performance of Portable Microbial Impactors. Aerosol
Science and Technology 44(1): 75-82. [0013] Tseng, C. C. and C. S.
Li 2005. Collection efficiencies of aerosol samplers for
virus-containing aerosols. Journal of Aerosol Science 36(5-6):
593-607. [0014] Verreault, D., S. Moineau and C. Duchaine 2008.
Methods for sampling of airborne viruses. Microbiology and
Molecular Biology Reviews 72(3): 413-444. [0015] Wang, Z., T.
Reponen, S. A. Grinshpun, R. L. Gorny and K. Willeke 2001. Effect
of sampling time and air humidity on the bioefficiency of filter
samplers for bioaerosol collection. Journal of Aerosol Science
32(5): 661-674.
[0016] The collection of bioaerosols is currently performed by a
number of devices that have been available for quite some time.
Common bioaerosol sampling devices include: [0017] Impactors where
a jet of air deposits the bioaerosol particle on a media surface.
[0018] Impingers where the jet of air impinges on a surface within
a liquid filled container. [0019] Filters where the particles are
collected on the surface of the filter.
[0020] Impactors are limited with respect to sampling time because
the collection media used to enumerate the number of colonies of
organisms for viability after collection is subject to desiccation,
thus limiting the sampling time. Also typical impactors designed
for microorganisms have a lower particle size collection limit of
about 0.5 micrometers. (Anderson, A. A. (1958) New sampler for
collection, sizing, and enumeration of viable airborne particles,
J. Bacteriol. 76, 471-484)
[0021] Impingers are limited in their sampling time from the
evaporation of the collecting fluid. The collection efficiency is
dependent on the volume of fluid in the impinger. Also the
microorganisms may be lost by reaerosolization from the fluid
during sampling (Grinshpun, S. A., K. Willeke, V. Ulevicius, A.
Juozaitis, S. Terzieva, J. Gonnelly, G. N. Stelma and K. P. Brenner
(1997) Effect of impaction, bounce and reaerosolization on the
collection efficiency of impingers. Aerosol Sci. Technol. 26,
326-342.).
[0022] Filters and other collection media such as membranes have
long been used to trap aerosol and bioaerosols for subsequent
analysis thereof. Filters with a poor figure of merit or quality at
least require higher pressures to force air flow through. An
example consequence is that in portable samplers operation is
severely limited due to battery life in the samplers with filters
with high pressure drop. Filter figure of merit or quality is
defined as FoM=-log (Pt)/.DELTA.P, where Pt is the penetration of
particle at a specific size through the filter and .DELTA.P is the
pressure drop at a specific gas flow rate. The larger the FoM, the
better will be the performance of the filter. See Hinds, W. C.
(1982) Aerosol Technology, Wiley, New York, N.Y.). Further, the
flow of air through the filters or membranes after a biological
aerosol has been trapped can lead to the desiccation of the medium
about the bioaerosol and death of the bioaerosol.
[0023] Thus, in general, a list of existing air sampling
technologies for bioparticles and their drawbacks are provided
below.
TABLE-US-00001 Typical longest Sampler d.sub.50 sampling time Notes
Impinger ~0.3 .mu.m 30 min Good for short e.g. AGI-30 term sampling
Impactor ~0.7 .mu.m 20 min Collection on e.g. Anderson agar reduces
desiccation SKC ~0.3 .mu.m 8 hrs Fluid for long BioSampler term
sampling interferes with PCR Filtration * 60 min.sup..dagger.
Desiccation is a e.g. 37-mm significant cassette with problem with
Nucleopore filtration * Filtration has a most penetrating size
about 0.1 to 0.3 .mu.m with efficiency of collection typically high
(>80%) across size range. .sup..dagger.Longer term sampling is
possible but organisms do not survive.
SUMMARY OF THE INVENTION
[0024] In one embodiment of the invention, there is provided a
bioparticle collection device including a collection medium
including a plurality of fibers formed into a fiber mat and
configured to collect bioparticles thereon, and includes a
viability enhancing material provider disposed in a vicinity of the
plurality of the fibers and configured to provide a viability
enhancing material to the collected bioparticles to maintain
viability of the bioparticles collected by the fiber mat.
[0025] In one embodiment of the invention, there is provided an
aerosol collection system including an aerosol pumping device
configured to entrain particles in a gas stream, an aerosol
saturation device configured to saturate the particles in the gas
stream with a biocompatible liquid or vapor and an aerosol
collection medium downstream from the aerosol saturation device.
The aerosol collection medium includes a plurality of fibers formed
into a fiber mat for collection of the saturated aerosol particles
and a viability enhancing material provider disposed in a vicinity
of the plurality of fibers and configured to provide a viability
enhancing material to the collected bioparticles to maintain
viability of the bioparticles collected by the fiber mat.
[0026] In one embodiment of the invention, there is provided a
method for collecting aerosols. The method entrains particles in an
gas stream, saturates the particles in the gas stream with a
biocompatible liquid, and collects the saturated aerosol particles
in a filtration medium including a plurality of fibers formed into
a fiber mat and a viability enhancing material provider disposed in
a vicinity of the plurality of fibers and configured to provide a
viability enhancing material to the collected bioparticles to
maintain viability of the bioparticles collected by the fiber
mat.
[0027] In one embodiment of the invention, there is provided a
bioparticle collection device including a collection medium
including a plurality of fibers formed into a fiber mat and an
osmotic material disposed in contact with the plurality of
fibers.
[0028] In one embodiment of the invention, there is provided an
aerosol collection system including an aerosol pumping device
configured to entrain particles in a gas stream, an aerosol
saturation device configured to saturate the particles in the gas
stream with a biocompatible liquid, and an aerosol collection
medium downstream from the aerosol saturation device. The aerosol
collection medium includes a plurality of fibers formed into a
fiber mat for collection of the saturated aerosol particles and an
osmotic material disposed in contact with the plurality of
fibers.
[0029] In one embodiment of the invention, there is provided a
method for collecting aerosols. The method entrains particles in a
gas stream, saturates the particles in the gas stream with a
biocompatible liquid, and collects the saturated aerosol particles
in a filtration medium including a plurality of fibers formed into
a fiber mat, and an osmotic material disposed in contact with the
plurality of fibers.
[0030] It is to be understood that both the foregoing general
description of the invention and the following detailed description
are exemplary, but are not restrictive of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] A more complete appreciation of the invention and many of
the attendant advantages thereof will be readily obtained as the
same becomes better understood by reference to the following
detailed description when considered in connection with the
accompanying drawings, wherein:
[0032] FIG. 1 is a table showing sampling challenges for the
sampling and preservation of bioaerosols;
[0033] FIGS. 2A and 2B are schematics of fiber structures of the
invention;
[0034] FIG. 3 is a schematic of a condensation growth tube with
impaction of droplet encapsulated particles on a fiber substrate of
the invention;
[0035] FIG. 4 is a schematic of a combination of the condensation
growth tube and a fiber filter where the particles are removed by
filtration;
[0036] FIG. 5 is a schematic showing the collection of bioaerosol
particles in a nanofiber filter;
[0037] FIG. 6 is a schematic showing a combination of a humidifying
section followed by a fiber filter of the invention;
[0038] FIG. 7 is a schematic showing the fiber filter with
injection of water into the fiber filter to maintain an environment
on the filter of 70% RH in this one illustrative example;
[0039] FIG. 8 is a schematic of applying fiber collection surfaces
to bioaerosol collection with an impactor;
[0040] FIG. 9 is a schematic of the combination of humidification
of the bioaerosol with an impactor containing fibers on the
collection surface;
[0041] FIG. 10 is a schematic showing a cascade impactor with fiber
collection surfaces and water introduction to maintain a controlled
relative humidity;
[0042] FIG. 11A is Table 1 depicting an example of polymer and
surface chemistries studied in this invention;
[0043] FIG. 11B is a SEM micrograph of a perspective view of a
nanofiber structure formed by deposition of PU on a part of a web
material;
[0044] FIG. 11C is another SEM micrograph of a perspective view of
a nanofiber structure formed by deposition of PU on web material
showing nanofiber coverage and orientation over an opening in the
underlying web material;
[0045] FIG. 11D is another SEM micrograph showing a cross section
of a nanofiber structure formed by deposition of PU on web
material;
[0046] FIG. 11E is another SEM micrograph of a nanofiber structure
formed by deposition of PU on web material;
[0047] FIG. 11F is a composite view showing differently scaled
depictions of a nanofiber sampling filter in a 37 mm cassette
format;
[0048] FIG. 12A is a composite of two scanning electron micrograph
SEM images showing a collection including a Bacillus globigii (Bg)
spore and what are likely MS2 virus particles;
[0049] FIG. 12B is a graph of pressure drop curves (pressure drop
versus face velocity) for two common commercial air sampling filter
materials as compared with nanofiber filter media composed of PSU
or PU deposited thereon;
[0050] FIG. 13 is a bar graph depicting colony forming units per
liter of air sampled for a challenge of Bacillus for different
sampler configurations and substrates;
[0051] FIG. 14 is a bar graph depicting colony forming units per
liter of air samples for Pseudomonas for different sampler
configurations and substrates;
[0052] FIG. 15 is a bar graph depicting plaque forming units per
liter of air samples for bacteriophage MS2 for different sampler
configurations and substrates;
[0053] FIG. 16 is Table 2 depicting the results of the collection
of Bg CFUs and MS2 PFUs by condensation growth tube (CGT) with
impaction onto PU nanofibers, the All-Glass Impinger (AGI), and an
aerodynamic particle sizer (APS);
[0054] FIG. 17 is Table 3 depicting a comparison of the fiber
filter mats of the invention to a standard Teflon filter using the
virus MS2;
[0055] FIG. 18 is a comparison of viabilities between fiber filter
mats of the invention and gelatin and Teflon filters where a
bioaerosol of Serratia was sampled for 3 hours;
[0056] FIG. 19 is a comparison of viabilities between fiber filter
mats of the invention and gelatin where a bioaerosol of Serratia
was sampled, and shows the impact of sampling face velocity on the
viability of Serratia;
[0057] FIG. 20 is a depiction showing of the viabilities obtained
when collecting fragile Yersinia rohdei using RH controlled
filtration with the fiber filter mats of the invention;
[0058] FIGS. 21A and 21B are Tables 4 and 5 depicting organism
survivability on different surfaces and different environmental
conditions;
[0059] FIG. 22 is a depiction showing the storage of the slightly
fragile Staphylococcus and very fragile organism Yersinia at
different storage conditions;
[0060] FIG. 23 is a schematic depiction of a sample storage device
incorporating a moisture providing material; and
[0061] FIG. 24 is a schematic depiction of another sample storage
device incorporating a moisture providing mechanism.
DETAILED DESCRIPTION OF THE INVENTION
[0062] As used herein, "bioparticles" means microbes and other
biological particles such as for example bacteria, viruses, and
biologically derived particles such as proteins, cell fragments,
etc.
[0063] As used herein, "viable" or "viability" is defined as the
capability of having a collected organism becoming active again
after being placed into a favorable environment. For example, a
collected bacteria spore or vegetative bacterium being placed into
a growth media and incubated under appropriate conditions for
growth resulting in growth and reproduction of the organism. For
example, a collected virus being exposed to its desired host and
incubated under appropriate conditions resulting in the virus
infecting the host.
[0064] As used herein, "collection viability" means the capability
to keep a percentage of bioparticles in a collection medium of this
invention alive during the collection event.
[0065] As used herein, "storage viability" means the capability to
keep a percentage of bioparticles in a collection medium of this
invention alive from the time of collection until the bioparticles
are analyzed or counted.
[0066] As used herein, "viability enhancement" or "enhanced
viability" encompasses both collection viability and storage
viability and means the capability to collect a percentage of
bioparticles from a medium without death and keep the collected
bioparticles alive until the bioparticles are analyzed or
counted.
[0067] As used herein, an "osmotic material" is as any material
that has the capacity to provide transport of liquids (such as for
example water or nutrients) to or from the collected bioparticles.
For example, a fiber composed of a hydrophilic polymer would
represent on one kind of osmotic material.
[0068] As used herein, "design limiting" organisms are organisms
which are extremely fragile and extremely difficult to keep
alive.
[0069] In order to determine if an organism is infectious for the
purpose of making health related decisions, the viability must be
assessed by culture methods where the presence of live organisms at
the start of the culture is needed.
[0070] Maintaining viability during and after collection is well
known to be a challenge. Some organisms are very hardy, such as
bacterial spores. These organisms can be very difficult to kill.
The same traits that make them difficult to kill make them more
readily kept alive or viable during collection and storage. Other
organisms are extremely fragile and extremely difficult to keep
alive. Maintaining viability of these design limiting
microorganisms during collection and during storage is a challenge.
Organisms lose viability during collection due to desiccation by
either the air moving past these organisms during the collection
process or from a process such as evaporation. Also, any condition
that leads to an increase in hydroxyl radicals will decrease
viability.
[0071] Thus, collection of bacteria and virus (microbes) while
keeping these bioparticles viable in the case of long term sampling
is problematic.
[0072] Referring now to the drawings, wherein like reference
numerals designate identical or corresponding parts throughout the
several views, FIG. 1 details the challenges overcome by the
invention for representative organisms important from a health
standpoint. More specific, challenges determined by the inventors
for conducting long term air sampling, but are not restricted to
long term air sampling, in the collection of viable bioparticles
include: [0073] Viability during sampling [0074] Sampling duration
time [0075] Viability of collected sample during storage [0076]
Compatibility of collected sample on the collection medium with
analysis techniques.
Bioparticle Sample Collection Devices
[0077] Electrospun micro and nanofibers from polymer solutions
provide a high surface area environment with tunable surface
chemistries which can be conducive to the collection and retention
of biological particles. Indeed, the invention in one embodiment
provides a sampling device for the collection and recovery of
particles, including biological particles such as bacteria,
viruses, and yeasts. The sampling device provides for enhanced
viability of biological particles and provides for quantitative
recovery of samples for laboratory analysis, as detailed below.
[0078] The invention provides for a device, based on a fiber mat or
a nanofiber mat that provides for collection of bioparticles
including bacteria, fungi, viruses, and other biological particles
(e.g., bioaerosols). The collection is achieved in one aspect of
the invention either through the use of the fiber mat as a filter,
for example a high efficiency low pressure drop nanofiber
flow-through filter, through the use of the fiber mat as a
substrate for impaction of particles, or for the use of a fiber mat
as a wipe.
[0079] In one embodiment of the invention, the bioparticles are
kept viable for extended periods of time (e.g., 1 day to >7
days) without extraordinary efforts because the biological
particles are collected in a moisture-rich (or nutrient-rich) fiber
mat or nanofiber filter mat. Furthermore, samples can be recovered
from the mats for analysis by extraction in buffer or other
suitable liquid. Alternately, the fibers can be configured to be
dissolved using, for example a low acid or enzymatic solution.
Indeed, in one embodiment of the invention, the nano or microfiber
material can be constructed from polymers that provide for
dissolution in water or an appropriate buffer. Such capability can
improve recovery of collected bioparticles for culture and
non-culture analysis method such as PCR (polymerase chain
reaction), ELISA (Enzyme-Linked Immunosorbent Assay), and a variety
of other molecular and biochemical techniques.
[0080] In one embodiment of the invention, the fibers are deposited
on a variety of backing materials which could include moisture
absorbing properties or ability to provide moisture to the fiber
mat; for example, super-soaker polymers, hydrophilic polyurethane
foam, blotter paper, polymer nonwoven mats containing hydroscopic
salts such as lithium chloride, and related methods. Accordingly,
the fibers of the invention can form in one embodiment a
bioparticle collection device including a collection medium
including a plurality of fibers formed into a fiber mat and an
osmotic material disposed in contact with the plurality of
fibers.
[0081] In one embodiment of the invention, the structure and
surface chemistry of the fibers, incorporation of additives, or
mixed fiber materials incorporating osmotic materials can
contribute to the collection and preservation of the bioparticles.
Furthermore the container or packaging of the fiber material can
aid in preservation. For example, a sealed container containing a
hydrogel or other material can be used to maintain RH to aid in
viability preservation.
[0082] U.S. Pat. No. 4,805,343 (the entire contents of which are
incorporated herein by reference) describes for example cellulose
acetate hollow fibers that have osmotic properties. Such fibers (or
other hydrophilic fibers) could be used in the present invention to
provide an external supply of water or nutrients transported to the
fiber mats collecting the bioaerosols. Alternatively, cellulose
acetate fibers could be intermixed into the fiber mats collecting
the bio-aerosols.
[0083] The use of a fibrous matrix to collect and preserve the
bioparticles also provides advantages from the equipment design and
operation point of view. A long term (>8 hrs) liquid-based
sampler typically would require a fluidics system to remove sample
and replenish buffers. An RH-controlled fibrous material format
would not require as an extensive fluidics system. Furthermore, if
a large amount of dust, pollen, and other small particles are
present in the fluidics system, then the instrument could become
clogged.
[0084] A fibrous matrix approach that is free of a fluidics system
could tolerate samples laden with dust, but these particles would
not shut the system down. Additionally, the mass of liquid needed
to operate a system long term could be significantly less. The
weight and complexity of a low liquid use or nearly liquid-free
sample collection/preservation system could also be much less
compared to a liquid collection system.
[0085] In one embodiment of the invention, the fibers are deposited
on various backing materials, and the combination of the fibers and
the backing materials is used as an impaction substrate for
collection of the aerosol. For example, the fibers can be
electrospun onto a foil and placed as a part of the impaction plate
in a standard impactor for air sampling.
[0086] The fiber matrix (and especially a nanofiber matrix)
provides a high surface area environment for collecting organisms.
At the micro-scale of bacteria and viruses, surface chemistry can
be important. Using polymers provides for adjustable surface
chemistries from hydrophilic to hydrophobic. Furthermore, hydrogels
including polymer networks that readily hold water can be used to
regulate the moisture content of the nanofiber matrix. Examples of
such systems include polymers of acrylic acid combined with sodium
hydroxide and co-polymers of poly(2-hydroxy ethyl methacrylate)
(polyHEMA). Complex multi-fiber and layered structures can easily
be fabricated to provide a mixed environment that cannot be
obtained with a liquid system. This mixed environment can
potentially provide a way for a variety of organisms that prefer
different environmental conditions to exist in the same sample. An
example of a mixed environment is simultaneously electrospinning
two different polymers onto a common collection substrate thus
creating a fibrous mat with two different polymers which would have
two different surface chemistries and/or fiber diameters.
[0087] In one embodiment of the invention, the fibrous matrix
sample collection device includes mechanisms such as those
described above or other mechanisms to provide moisture or to
maintain the RH in a desired range, for example from 65% to 85% or
more precisely 70% to 85% or more precisely 75% to 81%.
[0088] In one embodiment of the invention, a polyurethane PU fiber,
the structure of the PU nanofibers, the corresponding nonwoven, and
the RH all contribute to viability maintenance. In this embodiment,
the viability enhancing aspect appears to be only the PU nanofiber
mat and surface humidity of the nanofibers, and there is no need
for an additional osmotic material, although such an addition could
be used.
[0089] In one embodiment of the invention, the sample collection
device provides viable storage at ambient temperature and RH. In
another embodiment of the invention, viability maintenance is
enhanced, especially for particularly fragile organisms, via
storage at cooled conditions. Storage of fragile organisms such as
Yersinia has been demonstrated for more than 9 days when stored on
a polyurethane (PU) nanofiber media in a laboratory
refrigerator.
[0090] On one hand, while keeping collected organisms wet may
result in germination or growth, and the collection conditions
might be good for one class or organisms, the collection conditions
might be bad for another class of organisms. On the other hand, an
overly dry environment can also kill organisms. A fibrous matrix
(optionally combined with other humidity control devices) can
provide a relative humidity (RH)-controlled environment to improve
preservation of viability of bioaerosols while potentially
simplifying sample handling and storage.
Filter Collection Systems
[0091] Nanofibers can be used in one embodiment of the invention as
a low pressure drop, high efficiency collection filter in any
standard sampling form such as the commercial `37 mm air monitor
cassette` or other sampling cassette device. (The nanofibers can
also be used in an impaction device for example in an eight-stage
impactor.)
[0092] FIGS. 2A and 2B show schematics of various fiber structures.
The different fibers designations indicate different function in
microorganism preservation. In one embodiment, fibers are present
that contain or regulate water moisture. For example, the fibers
such as hydrogel polymers or crosslinked polyHEMA or gelatin or
similar such material can be used as for hydration. Alternate
versions include hydrophilic polymers, like cellulose and its
derivatives (e.g. cellulose acetate) and may include incorporation
of hydroscopic salts such as lithium chloride; or hydrogel
particles, such as those formed from acrylic acid combined with
sodium hydroxide, with these particles entrapped in the fiber
matrix. In FIGS. 2A and 2B, the white space between the two
designated types of fibers represents air space or other fibers in
the collection mat (for example having a density and size to
promote collection viability and storage viability of a
bioaerosol).
[0093] In one embodiment, the white space between the two
designated types of fibers may be filled with particles which
themselves contribute to the viability of the collected
bioaerosols. These particles can be introduced during the
electrospinning process in a manner as described in U.S. Pat. No.
7,297,305 (the entire contents of which are incorporated herein by
reference). For example, particles (e.g., antioxidant particles or
nutrient particles) which can contribute to the viability of the
collected bioaerosols can be introduced into the fluids suitable
for electrospraying and/or electrospinning. Alternatively, these
particles can be introduced in a manner as described in U.S. Pat.
Appl. No. 2006/0264140 (the entire contents of which are
incorporated herein by reference).
[0094] In this process, particles which can contribute to the
viability of the collected bioaerosols are delivered into a
fiber-extraction region of an electrospinning apparatus. The
introduced particles collide and combine with the electrospun fiber
material during formation of the fibers and the fiber mat.
Alternatively, these particles can be introduced after the
electrospinning process by flowing a solution (non-reactive with
the fibers in the fiber mat and containing the particles of
interest) through the fiber mat. The solution can be thereafter
evaporated or retained if the solution itself is a substance which
can contribute to the viability of the collected bioaerosols.
[0095] The fibers in FIGS. 2A and 2B may be aligned or may have
random orientations. The fibers in FIGS. 2A and 2B would in one
embodiment be in contact with one another in the fiber mat.
[0096] In one embodiment of the invention, the hydration fibers are
not be required. In one embodiment of the invention, the
preservation fibers are not required. When used, the preservation
fibers, due to their surface chemistry and structure, promote
preservation of the bioparticles. A more detailed description of
preserving fibers is provided below.
[0097] Accordingly, FIG. 2A illustrates intermixed fiber material
made by simultaneous electrospinning onto a common collection
plane, and FIG. 2B illustrates the concept of a layered structure
that can be formed either by sequential electrospinning to make a
layered structure or by spinning from opposing directions to a
common plane to simultaneously build to two sides of the composite,
layered structure.
[0098] As noted above, the fiber mat of the invention can be
configured as an impaction substrate or as a flow though filter,
and can be used in a variety of air sampling systems and
configurations.
Methods of Conditioning Bioparticle prior to Collection
[0099] In one aspect of the invention, the conditioning of inlet
air containing bioparticles facilitates the collection of viable
bioparticles. In one aspect of the invention, the collection of the
bioparticles occurs onto an appropriate substrate (media) that aids
in collection of viable bioparticles, aids in storage of the viable
bioparticles, and permits analysis via a variety of techniques
(e.g. live culture, PCR-based analysis methods, immuno-based
assays, etc.).
[0100] FIG. 3 is a schematic of a condensation growth tube with
impaction of droplet encapsulated particles on a fiber substrate of
the invention. The fiber substrate provides an environment for
preserving microorganism viability during sampling and storage.
U.S. Pat. No. 6,712,881 (the entire contents of which are
incorporated herein by reference) depicts a water condensation
growth tube (CGT) technology where particles are enlarged by water
condensation. The air containing bioparticles and other particles
flow through the condenser having a second temperature greater than
the flow temperature and a vapor pressure of condensing vapor at
the walls of the condenser near saturation. This technology is
capable of condensationally enlarging particles as small as viruses
(0.01 .mu.m-0.02 .mu.m) into 2-.mu.m diameter water droplets while
maintaining a laminar flow. After the droplets have been formed,
the encapsulated particles can be focused and collected inertially
at low velocities, reducing the potential damage to the
microorganism and minimizing the energy and noise associated with
the pump.
[0101] In one embodiment of the invention, exit jets from a CGT
device can impact bioaerosol or other aerosol particles onto a
fiber substrate of the invention. In one embodiment of the
invention, in the CGT configuration, small particles (e.g.,
sub-micron sized particles) are enlarged through condensational
growth while still airborne, essentially encapsulating each
particle within a micrometer-size (or larger) moisture droplet that
is readily collected at low velocity onto the fibrous or
nanofibrous mat.
[0102] FIG. 4 is a schematic of a combination of the condensation
growth tube and a fiber filter where the particles are removed by
filtration. In this arrangement, the condensation growth tube is
coupled with a fiber filter. The particles are removed by the
filtration mechanisms of impaction, interception, and diffusion. In
one embodiment of the invention, the exit gas from the CGT flows
through a fiber filter. In one embodiment of the invention, in the
CGT, small particles (e.g., sub-micron sized particles) are
enlarged through condensational growth while still airborne,
essentially encapsulating each particle within a micrometer-size
(or larger) moisture droplet that is readily collected.
[0103] Accordingly, in this technique of the invention, a
bioparticle is exposed to the vapor or a working fluid (for example
biocompatible fluids such as water) in a saturation chamber.
Subsequently, vapor condensation onto bioparticles is induced by
either adiabatic expansion or cooling in the condensing chamber, or
by mixing with a cooler airflow.
[0104] With the CGT, sub-micron particles (including bioparticles)
can be grown using a supersaturated vapor to a size where
collection on the fiber mats or nanofiber mats of the invention is
enhanced in terms of particle entrapment and bio-particle
survivability. Accordingly, in one embodiment of the invention, the
formed particle-water-condensate bioparticles are collected on the
collection medium of the invention.
[0105] FIG. 5 is a schematic showing the collection of bioaerosol
particles in a fiber filter. The collection in the fiber filter
occurs by interception, impaction, and diffusion. In one
embodiment, a nanofiber filter has low pressure drop and high
efficiency and creates an environment for preservation of
microorganisms.
[0106] FIG. 6 shows a fiber filter following a humidifying section
which controls the humidity at the fiber filter at a target value
or range, for example 50 to 85% RH. The humidification chamber (in
one embodiment) is disposed at the site of the mixing of humidified
air with the bioaerosol sample. The humidification chamber (in
another embodiment) is a chamber where water is introduced into the
air by wetted porous walls to maintain e.g., a relative humidity of
70 to 80% at the filter.
[0107] FIG. 7 shows the arrangement of introducing water into the
fiber filter to maintain an environment e.g. a relative humidity of
70 to 80% which preserves microorganisms during sampling. The
humidity would alternatively be maintained during storage.
[0108] FIG. 8 shows combination of the fiber with a cascade
impactor. The fibers, including nanofibers, prevent rebound of the
particles and can provide an environment to preserve the
microorganisms. The impactors have small holes forming jets of air
directing particles at the collection stage at a high velocity
(usually less than 0.3 Mach). The inertia of the particles causes
the particles to impact on the fibers.
[0109] FIG. 9 shows an impactor containing fiber collection on
collection stages with a humidification of the bioaerosol.
Humidification (in one embodiment) involves the mixing of the air
containing bioaerosol with moist air or (in another embodiment)
evaporation of water within the humidification chamber from wet
porous walls.
[0110] FIG. 10 shows the introduction of water into an impactor
with fiber on the collection surface to maintain a controlled
humidity e.g., at 70% RH.
[0111] Accordingly, in this invention, there are provided a number
of ways for conditioning of bioparticles prior to collection which
include use of a condensation growth tube, adding water moisture to
the sampled air stream, and regulating the relative humidity (RH)
of the sampled air stream. Addition of water moisture or regulation
of the RH can be achieved via a number of methods including use of
a wet walled tube to provide humidity to the sampled air,
atomization of water to provide humidity to the sampled air, mixing
a wet or dry air stream with sampled air stream to provide air
stream at target RH (wet air could be generated through bubbling
air through water, a wet walled tube, atomization of water, etc.),
and other ways to regulate the RH of a sampled air stream.
Methods of Making Fiber Substrates for Bioparticle Collection
[0112] Electrostatic spinning of polymer solutions to form micro
and nano diameter fibers, better known as electrospinning, is a
ready method to make nonwoven fibrous mats. In one embodiment of
this invention, electrospinning is used to make fibrous mats but
other methods of fabricating mats of micro and nanofibers may also
be a route to form fibrous structures described in this invention.
U.S. Pat. Nos. 5,494,616 and 6,520,425; and Badrossamy M R et al.,
Nano Letters 2010, 10(6):2257 both describe alternative techniques
applicable to the invention. The entire contents of these documents
are incorporated herein by reference.
[0113] A wide variety of polymers can be electrospun into fibers
including both synthetic polymers such as polystyrene and natural
polymers such as collagen and gelatin. Polymers offer hydrophobic
to hydrophilic surface properties including functionalities similar
to sugars or proteins. FIG. 11A shows Table 1 is an example of only
a limited number of polymer and surface chemistries that are
suitable for this invention.
[0114] In terms of the use of electrospun fibers for filter mats
suitable for the invention, U.S. Pat. Appl. Publ. No.
(2005/0224999), the entire contents of which are incorporated
herein by reference, describes the use of an electronegative gas to
facilitate the electrospinning process by the introduction, for
example, of carbon dioxide (CO.sub.2) around the spinning orifice
or emitter. Gases such as CO, CF.sub.4, N.sub.2O, CCl.sub.4,
CCl.sub.3F, CCl.sub.2F.sub.2 and other halogenated gases can be
introduced into the electrospinning environment. These
electronegative gases stabilize the Taylor cone formed by the
polymer jet as it comes off the needle, reduces corona discharge at
the needle, and reduces fiber diameter. Furthermore, spinning in a
controlled environment ensures less contamination of the fibers,
improves safety, and adds another dimension of control parameters
that can be used to fine-tune fiber formation.
[0115] An electronegative gas can be passed coaxially with the
spinning needle along with use of a controlled gas environment.
Typically, a gas shroud is used to provide the coaxial gas flow. A
typical shroud can be in the shape of an annulus having an outside
radius of about 0.48 cm and an inside radius of about 0.40 cm.
Insulating and metallic shroud members can be used. A variety of
geometries and sizes are possible; such as for example a circular
outside with a hexagonal inside being an additional geometry. In
the annular geometry, a distance from an exit end of the annulus
where gas is emitted to the tip of the electrospinning element can
range from flush (0 cm) to 8 cm; with a typical distance being
around 4 to 5 cm, and with the distance being 4.7 cm for the
detailed examples later.
[0116] Control of the electrospinning conditions has produced
polymer nanofibers with an average fiber diameter AFD of 100 nm and
less. Nanofibers less than 400 nm have been found to improve the
filtration properties of the resultant fiber when combined with
other elements of the invention.
[0117] Additives in the polymer solution can make a substantial
difference in fiber size and quality. Addition of trace amounts of
a salt or a surfactant increases the solution conductivity and
hence the charge accumulation at the tip of the electrospinning
element resulting in larger stretching forces applied to the
forming fiber, hence smaller diameter fibers. The surfactant also
reduces the surface tension of the polymer allowing for even
smaller fibers to be spun. Lithium salts, (for example, lithium
chloride and lithium triflate) or surfactants such as tetra butyl
ammonium chloride (TBAC) are suitable for the invention. Lithium
salt concentrations from 0.01 to 3 wt % are suitable for the
invention. Concentrations of TBAC of between 0.06 and 0.4 wt %,
were exemplary, although other concentrations are suitable.
[0118] Stainless steel extrusion tips from 0.15 mm to 0.59 mm
internal diameters (ID) are suitable for the invention. Larger and
smaller diameters may also be used. Teflon.TM. capillary tubes with
ID from 0.076 mm to 0.31 mm are suitable for the invention. Larger
and smaller diameters may also be used. Both types of orifices can
produce small fibers. For both orifices, low flow rates of the
polymer solution (e.g., 0.05 ml/hr) coupled with high voltage drops
typically resulted in the smallest fiber diameters (e.g., AFD less
than 100 nm). In both cases, the voltage was set to 22 kV to 30 kV
for a 17.8 cm to 25.4 cm gap (i.e., distance between emitter 2 and
mesh 7). Of note is that the voltage per electrospinning-gap is one
parameter determining the pulling strength; this gap also
determines a travel time thus partly determining fiber stretching
time.
[0119] Besides stainless steel and Teflon.TM. extrusion tips, in
the invention, other materials (provided the materials are
non-reactive with the substance being electrospun including any
solvent used in the electrospinning process) can be used such as
for example polymers, glass, ceramic, or metal extrusion tips.
[0120] The relative humidity RH of the electrospinning chamber
effects fiber morphology. For example, when using 21 wt % PSu
(M.sub.w.about.35,000 g/mol) in DMAc, a high RH (e.g., >65%)
resulted in fibers that have very few defects and smooth surfaces
but larger diameters. A defect in a fiber is in general seen as a
deviation from a smooth round fiber of long length. Defects thus
are beads on the fiber, variations in fiber diameter in the axial
direction, etc. A low RH (e.g., <13%,) resulted in smaller
fibers but more defects. Modestly low RH (e.g., 40% to 20%)
typically produced small fiber size with fewer defects.
[0121] A variety of mechanisms are suitable in the invention to
control the chamber RH such as placing materials that absorb (e.g.
calcium sulfate) or emit water moisture (e.g., hydrogels),
operating a small humidifier in the chamber, and adding moisture
into the process gas streams prior to introduction to the
electrospinning chamber. For example, positive results were
obtained by bubbling CO.sub.2 through deionized (DI) water and then
introducing the humidified CO.sub.2 gas into the chamber. In one
embodiment of the invention, two gas streams (e.g., one humidified
and one dry) are used to obtain a desired RH for the chamber and/or
for the gas jacket flowing over the electrospinning orifice.
[0122] The fiber diameter obtained in the invention is a function
of the polymer molecular weight, the polymer architecture, the
solvent or solvents, the concentration of polymer in the solvent
system, the additives and their concentration, the applied
electrospinning potential, the gap between the spinning orifice and
ground, the size and shape of the spinning orifice, the polymer
solution flow rate, the flow rate and composition of the process
gas that flows over the needle, the RH of the process gas, and the
partial pressure of the solvent(s).
[0123] Other embodiments of the invention could use different
polymer solvent systems and hence different electrospinning
conditions to obtain appropriate nanofibers. Furthermore, the same
polymer solvent systems could be combined with different
electrospinning conditions to create improved fibers or fibers
tailored for alternative applications. For example, the jacket of
CO.sub.2 gas flowing over the needle could contain solvent vapor in
order to lower the evaporation rate of the solvent(s) in the
polymer jet formed at the needle tip, thus increasing stretching
time of the polymer fiber. The partial pressure of the solvent can
also be modified via control of temperature, pressure, and mixture
of solvents. The solvent concentration as determined by a relative
concentration in the atmosphere is controlled to between 0 and
100%.
Filter Support Structures
[0124] In addition to obtaining nanofibers having few defects and a
close distribution in fiber diameter sizes, the construction of a
support and preparation of the surface of the support affect the
resultant fiber mat and the resultant filter properties. In one
embodiment of the invention, a macroscopic mesh provides adequate
support for the nanofibers to withstand the forces exerted on
filter mat during filtration and collection of biological medium.
The support mesh contributes minimally to pressure drop of the
resultant filter.
[0125] Filters formed with rigid meshes that contained 1.27 cm,
0.635 cm, or 0.159 cm (i.e., American Engineering standard sizes:
1/2'', 1/4'' and 1/16'' respectively) openings using copper, brass,
nickel, stainless steel, and aluminum metal are suitable for the
invention. Smaller sizes have also been found acceptable including
meshes with openings as small as 0.031 cm. Aluminum window screen
with openings about 1.2 mm.times.1.6 mm is also an acceptable
support. The surface of the metal mesh, especially for aluminum
meshes, was subjected to cleaning to remove dirt and oils followed
by washing the mesh in diluted sulfuric acid (10 to 20%
H.sub.2SO.sub.4 in DI water by volume) to remove resistive oxides
and impurities. This cleaning improved nanofiber dispersion and
adhesion. The deposited fibers may not be totally dried of the
solvent used to dissolve the polymers. In that state, the fibers
adhere to the rigid mesh and after tensioning after drying form a
mesh-fiber structure beneficial to reduce pressure drop and
increase collection efficiency. Any number of metals or metal
alloys, with openings of various shapes (square, rectangle,
circular, diamond, oblong and odd shaped), with openings ranging in
size from about 12.7 mm down to 1000 times the AFD can be used in
the invention.
[0126] Adhesion of the nanofibers or fibers to the support mesh can
be improved via the application of an adhesive to the mesh directly
prior to electrospinning. The adhesive typically is a slow drying
adhesive permitting the adhesive to be tacky (i.e., adhesive) when
electrospun fibers are deposited. Alternately, in another
embodiment, the wires (or components) of the mesh can be coated
with a very thin layer of polymer that has surface groups which
interact (van der Waals, hydrogen-bond, dipole, electrostatic
attraction, etc.) with the polymer fibers being deposited on the
mesh. One example system is a thin coating of poly(glycidyl
methacrylate) (PGMA) on nickel mesh with nanofibers of poly(methyl
methacrylate) (PMMA) deposited on the coated mesh. An alternate
embodiment of the invention uses cross linkable systems that are
polymerized after the fibers are deposited. Examples include
chitosan nanofibers crosslinked with glutaraldehyde and polyvinyl
acetate crosslinked with borax; also, deposition of nanofibers on
adhesives such as Norland's line of curable adhesives based on
mercapto-ester compounds. These surface coatings increase adherence
and adhesion of the nanofibers to the support.
[0127] The metal mesh can be replaced with metal foams such as
ERG's Duocel.TM. metal foams; for example, Aluminum Durocel with 20
pores per inch (PPI; alternately an average pore size of 1.27 mm).
Foams can also be made with copper, nickel, and various other
metallic as well as polymeric materials. Porosities ranging from 10
PPI (2.5 mm pores) to 40 PPI (0.064 mm pores) are acceptable for
the invention.
[0128] The support mesh can be composed of a plastic that is
conductive. For example polyester or nylon screen (or coarse
nonwoven polymer mesh) is coated with a conductive finish such as
gold, palladium, or various metal alloys. The coating process can
be achieved by any number of established arts including vacuum
deposition (e.g., sputter coating, evaporation deposition, and
chemical vapor deposition), and chrome plating of plastics.
Alternately, the mesh can be composed of conductive plastic that
obtains its conductivity via embedded conductive particles (carbon
nanotubes, metals etc.); or, any method to make plastic mesh
conductive, semi-conductive, or electrostatic dissipating.
[0129] A nonwoven support that is conductive or made conductive
(e.g., sputter coating etc., as mentioned above) or moistening with
a conductive fluid such as water can be used. The nonwoven support
can make a larger contribution to the pressure drop but may be
acceptable in certain applications. In certain embodiments, use of
woven scrim materials may also be acceptable for a base of a
bioparticle collection medium.
[0130] The structure of the electric fields between the emitter and
ground, which drives fiber deposition, are controlled, in part, by
the design of the filter frame holder. Furthermore, the potential
of the support mesh can be controlled by an electric field
pulsation device (i.e., a voltage limiter or discharge device or an
electric field applicator device). The electric field pulsation
device can be configured to pulse an electric field at the
collector at least once (or frequently) during electrospinning of
the fibers to discharge charge accumulated on the electrospun
fibers.
[0131] Electrospun fibers carry charge to the mesh which is
discharged frequently to ground by the voltage limiter device
acting in this example as an electric field pulsation device. The
resultant electric field is oriented in the direction of the
spinning fibers and dynamically modifies the structure of the
electric field, thereby imparting improved fiber and mat properties
(as measured by the FoM of the mat).
[0132] Filters having figures of merit greater than 20 kPa.sup.-1
for average fiber diameters of the nanofibers less than 200 nm and
filters having a FoM greater than 40 kPa.sup.-1 for average fiber
diameters of the nanofibers less than 100 nm have been also
realized and are suitable for this invention.
[0133] The thickness of the fiber mat can vary from about 0.25
.mu.m (250 nm) to 500 .mu.m or beyond if needed, where most filters
had an average mat thickness in the range of 2 to 5 microns. The
average mat thickness numbers represent the average thickness of
the total fiber mat in a filter. Alternately the mat thickness can
be defined as layers of fibers with the thickness including from 4
to 4000 layers where 4 to 400, or 5 to 100, or 5 to 15 layers were
typical in various embodiments.
[0134] The flexibility of electrospinning even allows mixed
polymers such as coaxial, mixed (blended) in same fiber, or
deposited as layered or intermixed fibers. In addition to polymer
chemistry and mixture of polymers, additives such as salts,
proteins, and other materials can be included in the fibers via a
variety of methods. These include direct incorporation in the
electrospinning solution, deposited or coated onto the surface of
the fibers during electrospinning using coaxial spinning,
electrospin-spray or co-spinning U.S. Pat. No. 7,592,277 (the
entire contents of which are incorporated herein by reference). An
alternative to including additives as a part of the spinning
process is to use a post-spinning process to coat the fibrous mat.
The fibers can be coated after the fibers are formed via depositing
the fibers into a liquid bath containing the additives or via dry
or wet coating of the fiber mat after it is produced. A variety of
these combinations is also possible.
[0135] Accordingly, the fibers and nanofibers produced by the
invention include, but are not limited to, acrylonitrile/butadiene
copolymer, cellulose, cellulose acetate, chitosan, collagen, DNA,
fibrinogen, fibronectin, nylon, poly(acrylic acid), poly(chloro
styrene), poly(dimethyl siloxane), poly(ether imide), poly(ether
sulfone), poly(ethyl acrylate), poly(ethyl vinyl acetate),
poly(ethyl-co-vinyl acetate), poly(ethylene oxide), poly(ethylene
terephthalate), poly(lactic acid-co-glycolic acid),
poly(methacrylic acid) salt, poly(methyl methacrylate), poly(methyl
styrene), poly(styrene sulfonic acid) salt, poly(styrene sulfonyl
fluoride), poly(styrene-co-acrylonitrile),
poly(styrene-co-butadiene), poly(styrene-co-divinyl benzene),
poly(vinyl acetate), poly(vinyl alcohol), poly(vinyl chloride),
poly(vinylidene fluoride), polyacrylamide, polyacrylonitrile,
polyamide, polyaniline, polybenzimidazole, polycaprolactone,
polycarbonate, poly(dimethylsiloxane-co-polyethyleneoxide),
poly(etheretherketone), polyurethane, polyethyleneimine, polyimide,
polyisoprene, polylactide, polypropylene, polystyrene, polysulfone,
polyurethane, poly(vinylpyrrolidone), poly(2-hydroxy ethyl
methacrylate) (PHEMA), gelatin, proteins, SEBS copolymer, silk
(natural or synthetically derived), and styrene/isoprene
copolymer.
[0136] Additionally, polymer blends can also be produced as long as
the two or more polymers are soluble in a common solvent or mixed
solvent system. A few examples would be: poly(vinylidene
fluoride)-blend-poly(methyl methacrylate),
polystyrene-blend-poly(vinylmethylether), poly(methyl
methacrylate)-blend-poly(ethyleneoxide), poly(hydroxypropyl
methacrylate)-blend poly(vinylpyrrolidone),
poly(hydroxybutyrate)-blend-poly(ethylene oxide), protein
blend-polyethyleneoxide, polylactide-blend-polyvinylpyrrolidone,
polystyrene-blend-polyester, polyester-blend-poly(hydroxyethyl
methacrylate), poly(ethylene oxide)-blend poly(methyl
methacrylate), poly(hydroxystyrene)-blend-poly(ethylene
oxide)).
[0137] Other embodiments of the invention include the use of
polymers that are pH and/or thermal responsive such that the fiber
mat can later be modified, respond to a change in environment, or
easily dissolved. Example polymers include the commercial pH
sensitive polymers know as Eudragit polymers as well as copolymers
of N-isopropyl acrylamide (NIPAM) and N-methyacryloy-L-Leucine
(MALEU) or (N,N-dimethylamino)ethyl methacrylate (DMAEMA). A
similar approach would be to use polymers that are easily degraded
with enzymes such as Chitosan which is degraded by Chitosanase and
cellulose which is degraded by .alpha.-cellulase. Combinations of
polymer systems could be used to tune the fiber filter mat
properties to the particular application.
[0138] Other embodiments of the invention introduce an agent to the
fibrous matrix to reduce oxygen toxicity to bioparticles collected
in the collection medium. Such agents can be enzymes to reduce
oxygen toxicity including catalase. Such agents can be fullerenes
and modified fullerenes that have antioxidant properties. In
general, substances with known antioxidant properties can be added
to the sample collection medium of the present invention to improve
viability of the collected bioparticles.
[0139] Accordingly, in one embodiment of the invention, fibrous
mats can be electrospun that provide favorable conditions for a
variety of organisms.
Working Examples of Fibrous Mats for Bioparticle Collection
[0140] A variety of polymer fiber mats were prepared for this
invention using electrospinning. The polymer solution was prepared
to a target concentration in a solvent system amenable to
electrospinning. The solution was placed in a 3 ml syringe fitted
with a 30 G blunt tipped needle and placed into a controlled gas
environment (see U.S. Pat. No. 7,297,305, the entire contents of
which are incorporated herein by reference) of carbon dioxide and a
relative humidity (RH) of 30% to 35%. A high voltage was provided
to the needle through an electrical lead to create a voltage
gradient of about 1 kV/cm. The polymer flow rate was determined by
the electrospinning solution viscosity and ranged between 0.05 to
0.1 ml/hr. The fibers are collected on a substrate depending upon
the manner in which the fiber mat was used.
[0141] For use as an impaction substrate, the collection substrate
was aluminum foil or Whatman filter paper; for use as a flow-though
filter, the collection substrate was a light weight nonwoven filter
material such as Fiberweb Reemay style 2011, which is a nonwoven
filter material with a basis weight of 25.5 g/m.sup.2 and an air
permeability of 5,650 L/m.sup.2/s. In some cases, the nonwoven
backing is first coated with graphite, such as by spray painting
using Aerosdag G to enhance fiber adhesion to the substrate. The
electrospun fiber mat was then rinsed with filtered DI water and
dried in a sterile environment overnight to remove any residual
solvents. In some cases, additives were coated onto the finished
mat in a post-processing step.
[0142] One fibrous material prepared was polysulfone (PSu; Udel
P3500 LCD by Solvay Advanced Polymers) dissolved in
dimethylacetimide (DMAc) at a concentration of 21 wt %. 0.2 wt %
tert-butyl ammonium chloride (TBAC) was then added to improve the
electrospinning of the solution and the final fiber morphology. The
solution was electrospun for 90 minutes at a flow rate of 0.05
ml/hr and voltage gradient of 1.6 kV/cm.
[0143] Another fibrous material prepared was polyurethane (PU;
Pellethane by
[0144] Lubrizol) dissolved in dimethlyformamide (DMF) at 13 wt %.
The solution was electrospun at 1.2 kV/cm and a flow rate of 0.1
ml/hr for 90 minutes. More specifically, Pellethane 2103-90 AE Nat
polyurethane (PU) made by Lubrizol, electrospun at about 13 wt % in
dimethylformamide (DMF) to form micro and/or nanofibers was
deposited on a backing material. After the fibers are deposited on
the backing, the fibrous matrix was flushed with DI water and
allowed to dry in a clean environment.
[0145] The backing material can be any number of woven or nonwoven
media such as spunbound polypropylene. One example is Reemy
spunbound polypropylene nonwoven made by Fiberweb. Media with air
resistance of 500 CFM/ft2 to 1,500 CFM/ft2 are useful but media
with air resistance beyond this range may also be useful. In some
cases, using a backing material that is conductive or static
dissipating is advantageous. For example, a nonwoven can be spray
coated with graphite or coated with conductive material using
liquid or gas based (e.g. chemical vapor deposition) techniques.
Also materials known in the art that are static dissipating though
any number of methods may be useful.
[0146] Another example of a nanofiber structure for viable
collection and preservation is PU electrospun onto Fiberweb Reemy
2250 that was coated with aerodag (graphite) before
electrospinning. The PU fibers have an average fiber diameter of
320 nm, are free of beads, and form a nonwoven mat that is a few
microns to 10 s of microns thick. Alternatively, the PU fibers can
have an average fiber diameter in the range of 100 nm to 280 nm.
Alternatively, the fibrous matrix can have PU beads created during
the electrospinning process that are 1.5 to 4.5 microns in
diameter. In one embodiment, the average fiber diameter is 260 nm,
and the PU beads are about 4 microns in diameter. These nanofiber
materials with beads about 10 times to 20 times the size of the
fibers provide lower pressure drop.
[0147] FIGS. 11B-11E show SEMs of one embodiment of the nanofiber
structure formed by deposition of PU on Fiberweb that was first
coated with graphite. In FIG. 11B, a section of backing material
without nanofibers and a section with nanofibers are shown. The
nanofibers are supported by the nonwoven backing material. The
nanofibers provide for collection of the bioparticles. In FIG. 11C,
the beaded structure of the nanofibers and that they oriented
between the supporting macrofibers of the conductive nonwoven they
are deposited on is evident. This combination of beading and fiber
orientation provides a structure that has lower pressure drop
compared to other materials. In FIG. 11D, an edge (cross sectional)
view of the nanofibers deposited upon the nonwoven backing is
shown. The nanofiber layer is 10 s to 100 s of microns thick, but
much thinner than the supporting nonwoven material. In FIG. 11E a
high magnification SEM image is shown that indicates the fiber and
bead structure. The beads are typically oblong and are 10 to 20
times the size of the fibers. The fibers are on the order of 220 to
280 nm while the beads are on the order of 3.8 to 4.8 microns.
[0148] Another fibrous matrix of this invention includes nylon
fibers prepared for example from a 15.3 wt % solution of nylon 6
(Sigma Aldrich) dissolved in formic acid. The solution was
electrospun at a voltage gradient of 1.6 kV/cm and a flow rate of
0.05 ml/hr for 90 minutes.
[0149] Another fibrous matrix of this invention includes
polycaprolactone (PCL; Sigma Aldrich, ca 43,000 M.sub.w) fibers
prepared from a mixed solvent system. The solvent was composed of
80% methylene chloride and 20% DMF. PCL was dissolved in the mixed
solvent to a concentration of 18 wt % and electrospun at 1.1 kV/cm
with a flow rate of 0.1 ml/hr for 90 minutes.
[0150] Another fibrous matrix of this invention includes
polystyrene (PS; Sigma Aldrich, ca 350,000 M.sub.w) fibers prepared
from 23 wt % PS in DMF and electrospun for example at 1.2 kV/cm and
a flow rate of 0.1 ml/hr for 90 minutes.
[0151] In one embodiment, a fiber-facilitator is used. With a
fiber-facilitator (e.g. high molecular weight PEO), nanocellulose
and related cellulosic materials can be incorporated in the fibrous
matrix to form fibers. In other words, to assist in incorporating
cellulose-based materials into fibers, a facilitator which is known
in the art (e.g., high molecular weight PEO) to help in the
formation of fibers, can be used.
[0152] One way according to this invention to impart improved
viability maintenance to the fibrous mat was to apply a solution
containing additives to the electrospun mat after it was made and
rinsed with DI water. The coated mat would then be allowed to dry
in a sterile environment. For example, a solution containing a
protein containing solution tryptic soy broth can be applied to the
mat and allowed to dry before use.
Working Examples of Flow-Through Filters for Collecting
Bioparticles
[0153] A sheet of nonwoven nanofiber filter media was prepared
using PSu as described above with the fibers being deposited on
graphite coated fiberweb support material. 37-mm circle filters
were punched out of the sheet of nanofiber filter media and
packaged into a standard 37-mm air sampling cassettes for
testing.
[0154] FIG. 11F shows a nanofiber sampling filter in the 37 mm
cassette format realized by this invention. The efficiency of the
filter cassette was measured with 300 nm KCl aerosol particles and
was found to be >99.9% and have a pressure drop of 167 Pa for a
face velocity of 5.3 cm/s. A similar filter cassette was exposed to
bioaerosol, Bacillus globigii (Bg) or MS2, and analyzed via SEM and
molecular biology techniques. Collection of Bg spores and MS2
particles was obtained. FIG. 12A shows SEM images of a collected Bg
spore and what are likely MS2 virus particles.
[0155] In one embodiment of the invention, the collection
efficiency of the fiber mat structure (sampling filter) formed from
a plurality of micro or nanofibers can be >80% and more
specifically >95% for particles 0.025 .mu.m to 10 .mu.m in
diameter for a flow rate of 25 L/min for a 25 mm sampling cassette.
While at the same time, the pressure drop (air resistance) of an
unloaded fibrous sampling filter is less than 20 inches of water,
and more specifically less than 12 inches of water.
[0156] FIG. 12B compares the pressure drop curves (pressure drop
versus face velocity) for two common commercial air sampling filter
materials with nanofiber filter media composed of PSU or PU
deposited on graphite coated fiberweb as described above. The
structure formed by the lightweight backing material, the small
fiber diameter, the partially oriented fibers, and beaded fibers
(exemplified by PU nanofibers) provides for significant reduction
in pressure drop. These significantly lower pressure drops of the
nanofiber filters translate into advantages for both operation and
equipment design. With a lower pressure drop across the filter it
is easier to maintain the target RH of the filter and therefore
improve viability maintenance of the collected bioparticles.
Furthermore, with lower pressure drop the pumps and electrical
requirements for an air sampling device are smaller and more cost
effective.
[0157] Remarkably, despite the efficiency >95% and pressure drop
less than 12 inches of water, the sampling filter is able to
withstand loading with particles until pressure drops greater than
80 inches of water, and even as high as 100 inches of water.
[0158] The fibrous sampling filter described above is able to
operate at collection humidities ranging from 10% to 98% with no
loss of filtering integrity. However for viability considerations,
it is preferably operated in the range of 70% to 85%.
[0159] While described here in relation to flow through sampling,
these fibrous sampling filters have application in the other sample
collection devices described herein.
Bioparticle Collection, Testing, and Evaluation
[0160] Viable microorganisms were generated to test the viable
collection of the samplers. Bioparticle generation was accomplished
though the use of a Collison nebulizer containing a suspension of
microorganism. The microorganisms may be suspended in various
nebulizing fluids depending upon the organisms and the scenario
being tested. Nebulizer fluids range from sterile water to trypic
soy broth with antifoam. The composition of the nebulizing fluids
is often selected to simulate the conditions of various
bioparticles in the environment as the usual application for
bioparticle samplers is to collect microorganism from the ambient
or indoor air.
[0161] A recognized standard in the art of bioparticle collection
is the All Glass Impinger (AGI). The AGI is designed to draw
aerosols through an inlet tube (e.g., a capillary tube) to form a
jet of the aerosols to be captured by a liquid medium of deionized
water or impinger fluid. The jet tip is typically positioned 30-mm
above the base of the impinger. The AGI relies on the inertial
impaction as a means for collection. However, loss of sampling
liquid through evaporation and re-aerosolization of droplets
containing virus often reduces collection efficiency of liquid
impingers.
[0162] The AGI provides collection into liquid for particles larger
than about 0.3 microns. Due to the wet collection, the majority of
sampled organisms are collected in a viable state. However, this
method can only be operated for a short period of time, about 30
minutes. Yet, the AGI is a recognized collection system used as a
point of comparison to the fibrous material collection devices of
this invention. Andersen biological impactors and the SKC
biosampler were also suitable. The liquid collection fluid is
diluted (when necessary) and then analyzed.
[0163] Sampling of a controlled air stream containing an aerosol of
a microbe at controlled concentration was conducted to compare
sampling methods. In some cases an AGI is run for 30 minutes in
parallel with the other sampling technology with the AGI being
considered the "gold standard" to compare viable sampling
collection against. For example for a specified test bacteria, 6%
of AGI means that the method collected 6% of the colony forming
units per liter of sampled air that the AGI collected
[0164] Meanwhile, the fibrous material collection devices of this
invention were suspended in sterile extraction fluid (e.g--water,
phosphate buffered saline, tryptic soy broth [TSB]), diluted and
analyzed.
[0165] For comparison, the analysis for culturable organisms
followed standard procedures where an aliquot of collection fluid,
extraction fluid, or a dilution of either, is plated on
microbiological media appropriate for the microorganisms collected.
The plated media were incubated at a temperature favorable for the
microorganism growth and enumerated when colonies (bacteria) or
plaques (viruses) are countable.
Evaluation of Collection Via Condensation Growth Tube Condition
[0166] The method of conditioning the sampled aerosol with a CGT
followed by impaction onto the nanofiber mats of this invention was
compared with CGT followed by impaction onto other substrates, with
filtration air sampling using various filter materials, and the
AGI.
[0167] In FIG. 13, two different aerosol challenges of Bg generated
at two different relative humidities (RH in the aerosol generation
device) are sampled either with a CGT followed by impaction or via
a 37 mm-diameter air sampling cassette containing different filter
media. The filtration sampling conducted here does not use
additional conditioning (i.e. RH regulation) of the sampled air.
The CGT used is an 8 L/min module and the impaction substrate was
either a 37-mm filter or impinger fluid. The impinger fluid can
range in composition from sterile water to TSB with antifoam.
[0168] Filtration based air sampling using PSU nanofibers
demonstrates better viable collection than a CGT impacting on
impinger fluid as shown in FIG. 13, Challenge level 1. The
nanofiber filter is providing excellent collection efficiency of
the hardy Bg organism demonstrating that it is a viable method of
air sampling for hardy organisms.
[0169] CGT conditioning of the air followed by impaction onto PSU
nanofibers demonstrates excellent viable collection compared to CGT
with impaction onto impinger fluid or filtration based air sampling
using the industry standard Teflon filter (Teflo) as shown in FIG.
13, Challenge level 2.
[0170] Collection and preservation of vegetative organisms without
growth is significantly more challenging than spores (e.g. Bacillus
spp.). A model organism for studying hazardous vegetative bacteria
is Pseudomonas fluorescens. Results for collecting aerosolized P.
fluorescens are shown in FIG. 14; sampling was done either with
conditioning with the CGT followed by impaction onto various
substrates or via air filtration without any conditioning of the
sampled air. Collection of the organism via filtration without
humidity control results in desiccation and the organism is not
able to survive. Viable collection of P. fluorescens via the 8
L/min CGT impaction onto nanofiber substrates has been demonstrated
in FIG. 14. In this example two types of PSU nanofibers mats
deposited on a sorbent backing were used, one without additives and
one with dried TSB as an additive. The results showed that
collection on nanofibers, as long as there is humidification at the
collection point, worked well.
[0171] Another threat to health is viruses. Virus particles have
the potential to be much smaller than bacteria since the individual
viruses can be as small as 25 nm (0.025 .mu.m). Collection and
preservation of these particles can be quite challenging. MS2
bacteriophage was used as virus simulant and collected using
nanofiber substrates as shown in FIG. 15; here again collection is
done either with a CGT or filtration without humidity control. The
results showed that nanofibers effectively collected the virus in
both the cases of use with a CGT to condition the bioparticles
prior to collection or as a sampling filter without conditioning of
the sampled air. Results shown in FIG. 15 are reported as plaque
forming units (PFUs) per liter of air based on MS2 infection of a
lawn of E. coli.
[0172] To further assess the suitability of the CGT and the fiber
technologies of the invention for viable bioaerosol collect MS2
(approximately 0.02 to 0.03 .mu.m in diameter) and Bg
(approximately 0.9 .mu.m in diameter) were nebulized and collected.
Collection was performed in parallel with an 8 L/min CGT using a
PSU nanofiber filter as an impaction substrate and an AGI. For the
Bg experiments, an aerodynamic particle sizer (APS; TSI inc.) was
also used.
[0173] FIG. 16 includes Table 2 presenting the results of the
collection of Bg CFUs and MS2 PFUs by CGT with nanofibers, the AGI,
and the total particle number concentrations of Bg measured with
the APS. The APS showed consistent size distributions among runs,
with a geometric mean aerodynamic diameter of D.sub.gm=0.85 .mu.m,
equal to that expected for Bg. With a CGT sampler with impaction
onto nanofibers, the number of CFUs matched the concentrations
measured with the APS. Moreover, as shown in Table 4, the standard
deviation among runs, 18%, compared favorably to the 7% variability
in the APS counts. The AGI sometimes matched the APS counts and
sometimes yielded lower values.
[0174] Collection of the virus simulant MS2 is compared to the AGI
because it is one of the few bioaerosol samplers that are even
capable of efficiently collecting at sizes near 0.3 .mu.m. With CGT
and impaction onto nanofibers, the average concentration of
culturable PFUs was 34% higher than measured with the AGI. The
coefficient of variation was 16%. The absolute viable collection
efficiency is not yet known because the AGI is not efficient at
collecting 0.3 .mu.m and below, which is the size of individual or
small clumps of viruses. In short, the CGT with impaction onto
nanofibers is a better viable collector for virus particles than
the AGI.
[0175] Conditioning of the sampled air with a CGT followed by
impaction onto a nanofiber mat provides viable collection for
bacteria that is comparable to an AGI, However as moisture for the
CGT is continually fed to the system, sampling is no longer limited
to only 30 minutes required by the AGI. The CGT can perform very
long term sampling limited only by the size of the water reservoir
feeding the CGT and by the time the bioparticles can survive on the
collection substrate. This viability during storage is further
discussed below in the Section Storage of Collected Bioparticles.
Furthermore the CGT with impaction onto nanofibers has better
viable collection of virus particles compared to the AGI.
Evaluation of Collection Via Filtration and Humidity Controlled
Filtration
[0176] The methods sampling of air using filtration and of adding
moisture or controlling the humidity of the sampled air followed by
filtration were evaluated using aerosols of bioparticles and
comparison with industry standard filtration sampling methods or
the AGI.
[0177] FIG. 17 includes Table 3 showing a comparison sampling the
virus MS2 using filtration without humidity control of the sampled
air. The nanofiber filter mats of the invention are compared to a
standard Teflon filter. Table 5 assesses both collection efficiency
and viability. The collection efficiency of the polysulfone-based
nanofiber filter was noticeably higher than the standard Teflon
filter or the polystyrene nanofiber filter. (These results are for
materials not optimized for a specific microbe collection.)
[0178] For demonstration of humidity controlled filtration, a
method of controlling the humidity of the sampled air was
constructed that measured the RH immediately downstream of the
sampling filter. The RH of the sampled air was controlled via
mixing with a moist air stream, similar to that shown in FIG. 6.
The moist airstream was generated by passing clean, dry air through
a bubbler containing deionized water followed by HEPA filtration of
the humidified air. The ratio of sampled air to wet air was set at
the beginning of the experiment to provide target RH at the filter.
This ratio was noted and used to determine the actual volume of air
with aerosol sampled from the test chamber containing the
aerosolized bioparticles. Various filter types including nanofiber
filters of this invention were used. In the art, gelatin filters
are recognized as having the best viable collection of commercially
available materials. However, when used on their own, as is typical
in the art, they too are very limited in the duration time for
viable sampling of bioparticles, about 30 to 60 minutes.
[0179] Humidity controlled filtration was performed with the
vegetative bacterium Serratia using nanofiber filters of this
invention compared to gelatin or Teflon filters. The nanofiber mats
were punched into 25-mm circular filters and placed into a standard
25-mm air sampling cassette. The gelatin and Teflon filters were
used as received in a 37-mm air sampling cassette. Bioaerosol was
sampled for 3 hours and the results of CFUs of Serratia determined.
FIG. 18 compares the results of this experiment. The nanofibers and
gelatin perform better than the Teflon filters. It should be noted
that the gelatin filters have much higher pressure drop than the
nanofiber filters, see FIG. 12B.
[0180] A similar experiment to that shown in FIG. 12B was attempted
with longer term sampling to compare gelatin and PU nanofiber
filters for very long sampling times. However, the gelatin filters
deteriorated sometime after 3 hours of sampling and collected
bioparticles could not be recovered. The PU nanofiber filters were
found to withstand sampling times of more than 32 hours of
operation.
[0181] The impact of sampling face velocity as a function of filter
material for RH controlled filtration was tested with 30 minute
sampling of Serratia as shown in FIG. 19. PU nanofibers and gelatin
filters were used with an RH of 75% and the face velocity varied.
The PU nanofiber filter is able to collect viable bioparticles at
very high face velocities that actually result in rupture of the
gelatin filter. As expected the percent viable collected increases
with face velocity as more organism per area of filter are
collected at the higher flow rates. Operation at high flow rates,
as high as 100 L/min or even higher, is desirable for air
monitoring. For example release of biological weapon could result
in low concentrations of the organism in the air such that sampling
as much air as possible to generate as much collected organism as
possible is desired (that is as much single collected for the event
as possible).
[0182] To demonstrate collection of a very fragile vegetative
bacterium that is particularly difficult to collect in viable form
using filtration, Yersinia rohdei was collected using RH controlled
filtration with PU nanofiber filters compared to an AGI. FIG. 20
shows that indeed collection of even this very fragile organism is
possible and repeatable.
[0183] RH controlled filtration using nanofibers is an effective
way to perform long term viable collection of bioparticles. Using
the fibrous material collection devices of this invention provides
viable collection similar to gelatin when both substrates are used
in the same sampling system and same RH for short periods of time
and modest flow rates (face velocity less than 4,500 cm/min).
However, the fibrous material collection devices of this invention
provide several advantages over gelatin filters: 1) filter pressure
drop for nanofibers is much lower for the fibrous material
collection devices than gelatin; 2) the robustness of nanofibers in
the fibrous material collection devices is much greater than
gelatin. The fibrous material collection devices are able to
withstand high pressure drops, in excess of 100 inch-H.sub.2O, are
able to withstand long term operation at >75% RH (e.g., more
than 3 hrs, more than 24 hrs). Furthermore, the nanofiber filters
are free of contaminants that would interfere with or give false
results for microbiology assays.
Storage of Collected Viable Bioparticles
[0184] After collection, preserving the organisms is a significant
challenge, particularly in the case where samples are not
refrigerated.
[0185] In one embodiment of the invention, there is provided an
automated system for sequential particle collection and storage. In
one embodiment of the invention, several days of samples are stored
in a single sampling cassette that will also contain an electronic
tag indicating individual sample collection times, location, air
sampling volumes, and quality assurance (QA) parameters (e.g.,
flows, operating temperatures, water levels, and other performance
parameters). Another cassette can include consumables, including
water for the CGT operation or provision of elevated RH by other
method and any supplies for sample collection and preservation,
depending upon the collection and handling scheme selected. In one
embodiment, these cassettes would be exchanged in the field during
operation of the sample collector.
[0186] As a demonstration, organisms were inoculated via pipetting
solution containing microbes onto samples of various nanofiber or
fiber substrates and other substrates compatible with air samplers.
Two types of inoculations were done to simulate different
environmental conditions: "lightly protected" where a buffer with
0.25% TSB in sterile water was used, and "well protected" where a
full strength TSB buffer was used. When an aerosol containing
microorganisms or other bioparticles is generated in the natural
world, it always has other materials with it such as proteins,
sugars, sputum, dirt etc that provides protection for the
organisms. In a bioterrorist act, the bioparticles would be
purposely mixed with protective materials like protein. The samples
in this demonstration were stored at ambient temperature
(approximately 23.degree. C.) in controlled RH static chamber
tests. The relative humidity RH in this demonstration was
controlled via saturated salt solutions in the sealed chambers.
After storage, samples were extracted in buffer, such as TSB or
phosphate buffered saline, and platted on appropriate nutrient
media and incubated and organisms enumerated.
[0187] FIGS. 21A and 21B include Tables 4 and 5 showing the results
of survivability of model organism on various materials. "Alive"
means the organism was detected via live culture techniques and
"dead" means none were detected. A "D" and number indicated by days
of live detection, e.g. D7 means live culture detected at day
7.
[0188] In another set of experiments where PU nanofiber filters of
this invention were further studied, model organisms were
inoculated via pipetting and samples stored under various
conditions as shown in FIG. 22. The log change from the day of
inoculation (day 0) is reported. Storage of the slightly fragile
Staphylococcus is possible under a variety of conditions. The very
fragile organism Yersinia requires storage at cooled temperatures
such as 4.degree. C.
[0189] Storage of a range of bioparticles on nanofibers is
possible. For organisms that are hardy to moderately hardy, storage
under conditions not requiring cooling is possible. In some cases
storage under humidity controlled conditions such as those provided
by the RH control system of the sampled air are sufficient to
preserve the collected bioparticles. In the case of fragile and
very fragile organisms, cooled storage is required if viability
maintenance for more than a day or two are required. As
demonstrated, different collection substrates provide viable
maintenance for different organisms. With the flexibility of
electrospinning and other arts of making fibrous media a mixed
polymer fiber environment can be created to provide for viable
storage of a broad range of organisms not possible with a single
traditional material.
[0190] Prior to the invention, the best filter for collection
viability that currently existed was a gelatin filter. However, the
gelatin filter has a number of problems including contamination and
excessive drying during long term sampling without RH control,
which both negatively impact the storage viability. Another common
filter medium is PTFE (Teflon) filter. Yet, the results above,
especially for the design limiting organisms, show that both
collection and storage viability and the collection efficiency are
enhanced for the fibrous material collection devices of this
invention.
[0191] The above experiments with aerosolized bioparticles
demonstrated that nanofibers are good collectors of microbes
(bioparticles). The above experiments show that the selection of
polymer and fiber structure is one element impacting viability and
controllable by this invention. The above experiments show that
preventing desiccation of bioparticles is important is one element
impacting viability and controllable by this invention. The above
experiments show that viability maintenance can be achieved through
incorporation of viability sustaining additives, moisture, etc. and
by keeping the fibrous media in an RH regulated environment. These
aspects are controllable by this invention.
Alternative Applications
[0192] In addition to the collection of bioparticles, the various
embodiments of the sampler can collect other aerosol particles of
interest to the public health and air monitoring communities. The
sampler may be used outdoors to sample ambient air or for sampling
indoors in buildings, arenas, or transportation facilities. These
filters also offer an advantage because of their semi-transparency
for black carbon absorption analysis and low levels of analysis
interfering metals.
[0193] In these applications, the ambient air samples will contain
black carbon or soot from combustion sources, industrial pollution,
particles from atmospheric reactions, particles re-suspended from
soil and pavements, ocean generated particles and pollen, all of
which can be collected by the nanofiber collection devices of this
invention. When used for indoor applications, it is expected that
the occupant generated particles such as skin cells and residue of
personal care products, dust and fibers resuspended from carpets
and floors, smoking, and particles introduced from appliances such
as electrical motors and heaters or furnaces, and biological
material such as toxins and plant or animal debris, all of which
can be collected by the nanofiber collection devices of this
invention. Aerosol particles collected in the nanofiber filters
could be measured by light absorption or reflectance, microscopy,
weighing and chemical analysis.
[0194] While described above with respect to aerosol sampling, the
nanofiber media and aspects contributing to viable collection and
maintenance of bioaerosols have applications in the sampling of
bioparticles and organisms from surfaces and from water. For
example, a wipe or brush or other sample collection device
containing the nano or microfiber material described above that
provides sample collection and helps viability maintenance could be
used to collect bioparticles from a keyboard, lab bench, furniture,
vehicle interior, etc. The wipe or brush or other collection device
can then be transported with the viability maintenance materials to
a laboratory for analysis.
[0195] In one embodiment of the invention, the sample collection
device can be in the form of wipes, brushes, swabs, sorbent pads,
liquid filters, air filters, and/or similar devices for sampling
air, liquids, or surfaces. Applications include forensics,
regulatory compliance, surveillance, etc.
[0196] For embodiments of the invention where the nanofiber
material is used to collect microbes without incorporation of
mechanisms to control the humidity of inlet air, a container can be
used that provides a humidity controlled environment. For example a
wipe composed of a plurality of fibers with viability enhancing
properties for use in evidence collection. In one embodiment that
nanofiber wipe is stored in a sterile container with humidity
regulation. In other embodiments the wipe is stored sterile but
humidity is not required prior to use. The nanofiber wipe is then
used to collect a sample and is placed in the container where the
container provides a favorable RH environment for viability
maintenance during transport to the laboratory for analysis. The
container and wipe thus constitute a sample collection device that
provides for viability maintenance.
[0197] The samples can then be stored in a sample storage device
which can incorporate a moisture providing material or mechanism
such as a hydrogel, water saturated salt solution, water reservoir
separated from the nanofibers via a moisture permeable membrane
providing water transport, or a water reservoir connected to the
part of the container holding the nanofibers via a wick. Examples
of these are shown in FIGS. 23 and 24. FIG. 23 is a schematic
depiction of a sample storage device incorporating a moisture
providing material. FIG. 24 is a schematic depiction of a sample
storage device incorporating a moisture providing mechanism (e.g.,
a wick and a water reservoir). The moisture providing material and
the moisture providing mechanism can further provide nutrient or
antioxidants, as described above.
Generalized Aspects of the Invention
[0198] In one aspect of the invention, a collection device includes
a plurality of fibers formed into a fiber mat. The fiber mat is
configured to collect and maintain the viability of microbes and/or
bioparticles. The fibrous filter can be configured in any manner
used in air sampling/aerosol collection using a flow-through
filter. In one aspect of the invention, the fibers are configured
as an impaction substrate to collect and maintain microbes and/or
bioparticles for use in air sampling/aerosol collection using the
method of impaction. The fibrous filter can be configured in any
manner used for swabbing or the wiping of surfaces or the sampling
of bioparticles in liquids.
[0199] In one aspect of the invention, there is provided a
bioparticle collection device including a collection medium
including a plurality of fibers formed into a fiber mat and
configured to collect bioparticles thereon, and including a
viability enhancing material provider disposed in a vicinity of the
plurality of fibers and configured to provide a viability enhancing
material to the collected bioparticles to maintain viability of the
bioparticles collected by the fiber mat. In this aspect of the
invention, the viability enhancing material may or may not be a
subset of the plurality of fibers.
[0200] In one aspect of the invention, the filter or impaction
substrate includes a fibrous mat configured in terms of structure,
surface chemistry, and additives to provide enhanced support of
viability maintenance of the bioparticles collected.
[0201] In one aspect of the invention, a filtration or impaction
device for aerosol collection includes a fibrous mat and in
conjunction with a mechanism or method for conditioning the
moisture content of the air entering the air sampling device to a
value that provides enhanced collection and maintenance of the
bioparticles collected.
[0202] In one aspect of the invention, a device including a
condensation growth tube is used to increase aerosol particle size
with condensation of water moisture followed by impaction onto a
fibrous substrate or a subsequent filtration mechanism. The fibrous
substrate and/or the subsequent filtration mechanism provide a
collection mechanism of bioparticles and provide a mechanism for
maintenance of viability of the collected bioparticles.
[0203] In one aspect of the invention, the aerosol is exposed to
the vapor or a working fluid (such as for example water and other
fluids that are biocompatible, possibly including silicone fluids)
in a saturation chamber. Subsequently, vapor condensation onto
particles is induced by either adiabatic expansion or cooling in
the condensing chamber, or by mixing with a cooler airflow. The
enlarged particles are subsequently collected via impaction or
filtration on a nanofiber or fiber material.
[0204] In one aspect of the invention, a fibrous mat is configured
to provide enhanced recovery of the collected material. Enhanced
recovery includes 1) recovery of the particles such that the
extraction procedure does not decrease their viability; 2) a
collection and extraction which does not prevent or impede
subsequent analysis such as live culture, PCR-based techniques, or
any other chemical or physical analysis of the collected
material/organisms; and 3) enhanced release of the collected
material through dissolution of the fibrous material using select
solvents and/or processing conditions.
[0205] In one aspect of the invention, the fibers in the fibrous
material or the fiber mat have an average fiber diameter of less
than 10 .mu.m, or less than 1 .mu.m, or less than 500 nm, or less
than 300 nm, or less than 200 nm, or less than 100 nm.
[0206] In another aspect of the invention, there is provided a
method for collecting aerosols. This method includes entraining
particles in a gas stream, saturating the particles in the gas
stream with a solvent, and collecting the saturated aerosol
particles by a collection medium. The collection medium includes a
plurality of fibers formed into a fiber mat including and a
viability enhancing material provider disposed in a vicinity of the
plurality of fibers and configured to provide a viability enhancing
material to the collected bioparticles to maintain viability of the
bioparticles collected by the fiber mat.
[0207] This method also can inject the viability enhancing material
into the collection medium prior to collecting the aerosol
particles. The viability enhancing material injected can be at
least one of water, proteins, carbohydrates, sugars, salts,
phosphate buffered saline, and tryptic soy broth. This method also
can inject the viability enhancing material (such as those listed
above) into the collection medium during the collecting of the
aerosol particles. This method also can inject antioxidants such as
for example nitrous oxide (NO) into the collection medium.
[0208] This method also can introduce an agent to reduce oxygen
toxicity to the bioparticles collected in the collection medium.
Such an agent can include enzymes or fullerenes to reduce oxygen
toxicity.
[0209] In another aspect of the invention, there is provided a
bioparticle collection device including a collection medium
including a plurality of fibers formed into a fiber mat and an
osmotic material disposed in contact with the plurality of fibers.
The osmotic material can be a viability enhancing material
configured to maintain viability of bioparticles collected by the
fiber mat. The osmotic material can be a water-regulating material
configured to provide water to the fibers. The osmotic material can
constitute a nutrient supply providing nutrients to support
biological viability of biomaterial collected in the filtration
medium. The nutrient supply can be at least one of water, proteins,
sugars, carbohydrates, salts, phosphate buffered saline, and
tryptic soy broth.
[0210] The collection medium and the viability enhancing material
can be disposed in one of an air filter, a wipe, a brush, a swab, a
sorbent pad, or a liquid filter. The fibers can be made of
materials which are dissolvable in a bio-compatible solvent.
[0211] The collection medium can include a support (e.g., a rigid
support) supporting the collection medium. The support can be one
of a filter, a plastic foam, a metallic foam, a semi-conductive
foam, a woven material, a nonwoven material, a plastic screen, a
textile, and a high efficiency particulate air (HEPA) filter
medium.
[0212] In another aspect of the invention, there is provided an
aerosol collection system including an aerosol pumping device
configured to entrain particles in a gas stream, an aerosol
saturation device configured to saturate the particles in the gas
stream with a biocompatible liquid, and an aerosol collection
medium downstream from the aerosol saturation device. The aerosol
collection medium includes a plurality of fibers formed into a
fiber mat for collection of the saturated aerosol particles, and an
osmotic material disposed in contact with the plurality of
fibers.
[0213] The aerosol collection system can include a humidity control
device configured to maintain the collection medium at a relative
humidity from 50 to 100%, or at a relative humidity from 65 to 85%,
or at a relative humidity from 75 to 81%.
[0214] The aerosol collection medium in this aspect of the
invention can be at least one of a flow-through or an impaction
device. The osmotic material in this aspect of the invention can be
a viability enhancing material configured to maintain viability of
bioparticles collected by the fiber mat. The osmotic material in
this aspect of the invention can be a water-regulating material
configured to provide water to the fibers. The osmotic material in
this aspect of the invention can be a nutrient supply providing
nutrients to support biological viability of biomaterial collected
in the filtration medium. The nutrient supply in this aspect of the
invention can be a supply of at least one of proteins, sugars, and
salts.
[0215] The fibers in this aspect of the invention can be
nanofibers, can be formed of materials dissolvable in a
bio-compatible solvent. A support (rigid or not) can be used to
support the collection medium. The support in this aspect of the
invention can be at least one of a filter, a plastic foam, a
metallic foam, a semi-conductive foam, a woven material, a nonwoven
material, a plastic screen, a textile, and a high efficiency
particulate air (HEPA) filter medium.
[0216] In another aspect of the invention, there is provided a
method for collecting aerosols. The method included entraining
particles in an gas stream, saturating the particles in the gas
stream with a biocompatible liquid, and collecting the saturated
aerosol particles by a collection medium including a plurality of
fibers formed into a fiber mat including and an osmotic material
disposed in contact with the plurality of fibers.
[0217] This method also can inject the viability enhancing material
into the collection medium prior to collecting the aerosol
particles. The viability enhancing material injected can be at
least one of water, proteins, carbohydrates, sugars, salts,
phosphate buffered saline, and tryptic soy broth. This method also
can inject the viability enhancing material (such as those listed
above) into the collection medium during the collecting of the
aerosol particles. This method also can inject antioxidants such as
for example nitrous oxide (NO) into the collection medium.
[0218] This method also can introduce an agent to reduce oxygen
toxicity to the bioparticles collected in the collection medium.
Such an agent can include enzymes or fullerenes to reduce oxygen
toxicity.
[0219] In another aspect of the invention, there is provided a
bioparticle collection device including a collection medium
including a plurality of fibers formed into a fiber mat and an
osmotic material disposed in contact with the plurality of fibers.
The osmotic material can be a viability enhancing material
configured to maintain viability of bioparticles collected by the
fiber mat. The osmotic material can be a water-regulating material
configured to provide water to the fibers. The osmotic material can
constitute a nutrient supply providing nutrients to support
biological viability of biomaterial collected in the filtration
medium. The nutrient supply can be at least one of water, proteins,
sugars, carbohydrates, salts, phosphate buffered saline, and
tryptic soy broth.
[0220] The collection medium and the viability enhancing material
can be disposed in one of an air filter, a wipe, a brush, a swab, a
sorbent pad, or a liquid filter. The fibers can be made of
materials which are dissolvable in a bio-compatible solvent.
[0221] The bioparticle collection device can include a support
(e.g., a rigid support) supporting the collection medium. The
support can be one of a filter, a plastic foam, a metallic foam, a
semi-conductive foam, a woven material, a nonwoven material, a
plastic screen, a textile, and a high efficiency particulate air
(HEPA) filter medium.
[0222] Numerous modifications and variations of the invention are
possible in light of the above teachings. It is therefore to be
understood that within the scope of the appended claims, the
invention may be practiced otherwise than as specifically described
herein.
* * * * *