U.S. patent application number 17/103900 was filed with the patent office on 2021-05-20 for liquid to liquid biological particle fractionation and concentration.
The applicant listed for this patent is InnovaPrep LLC. Invention is credited to Alec Douglas Adolphson, David Scott Alburty, Steven Dale Graham, Zachary Allen Packingham, Andrew Edward Page.
Application Number | 20210148797 17/103900 |
Document ID | / |
Family ID | 1000005360742 |
Filed Date | 2021-05-20 |
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United States Patent
Application |
20210148797 |
Kind Code |
A1 |
Packingham; Zachary Allen ;
et al. |
May 20, 2021 |
LIQUID TO LIQUID BIOLOGICAL PARTICLE FRACTIONATION AND
CONCENTRATION
Abstract
The present disclosure provides for devices, systems and methods
for fractionation and concentration of particles from a fluid
sample. This includes a cartridge containing staged filters having
porous surface in series of decreasing pore size for capture of
particles from a fluid sample; and a permeate pressure source in
fluid communication with the cartridge; wherein the particles are
eluted from the porous surfaces and dispensed in a reduced fluid
volume.
Inventors: |
Packingham; Zachary Allen;
(Drexel, MO) ; Page; Andrew Edward; (Smithton,
MO) ; Alburty; David Scott; (Drexel, MO) ;
Graham; Steven Dale; (Overland Park, KS) ; Adolphson;
Alec Douglas; (Raymore, MO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
InnovaPrep LLC |
Drexel |
MO |
US |
|
|
Family ID: |
1000005360742 |
Appl. No.: |
17/103900 |
Filed: |
November 24, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
14058193 |
Oct 18, 2013 |
10845277 |
|
|
17103900 |
|
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|
|
61715451 |
Oct 18, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01D 63/088 20130101;
G01N 1/40 20130101; B01D 61/20 20130101; G01N 2001/4038 20130101;
B01D 63/082 20130101; B01D 61/22 20130101; B01D 2315/08 20130101;
G01N 1/4077 20130101; G01N 2001/4016 20130101; G01N 1/405 20130101;
G01N 1/4055 20130101; B01D 2321/02 20130101; G01N 2001/4088
20130101; B01D 2319/06 20130101 |
International
Class: |
G01N 1/40 20060101
G01N001/40; B01D 61/20 20060101 B01D061/20; B01D 61/22 20060101
B01D061/22; B01D 63/08 20060101 B01D063/08 |
Goverment Interests
GOVERNMENT INTERESTS
[0002] This subject disclosure was made with U.S. Government
support under Department of Homeland Security (DHS) Grant No.
D12PC00287. The government has certain rights in this subject
disclosure.
Claims
1. A device for fractionation and concentration of particles from a
fluid sample, the device comprising: a cartridge containing staged
filters having porous surface in series of decreasing pore size for
capture of particles from a fluid sample; and a permeate pressure
source in fluid communication with the cartridge; wherein the
particles are eluted from the porous surfaces and dispensed in a
reduced fluid volume.
2. The device in claim 1, further comprising a connecting portion
for connection to a concentrating unit.
3. The device in claim 1, wherein the filters are separated by a
small interstitial space.
4. The device in claim 1, wherein the membrane filters are
separated by a filter support with flow channel connecting the
permeate of one filter with the retentate of an adjacent smaller
pore filter.
5. The device in claim 1, wherein a sample is introduced into the
device perpendicular to a surface of each filter.
6. The device in claim 1, wherein valved fluidic connections
connect the interstitial space or flow channels between
filters.
7. The device in claim 1, wherein pneumatic, hydraulic, or
mechanical valves are integrated into the cartridge device.
8. The device in claim 1, wherein the filters are one or more of a
flat membrane filter, a flat ceramic filter, an affinity-based
filter, a flat depth filter, an electrostatically charged filter,
or a microsieve.
9. The device in claim 1, further comprising an elution buffer
distribution manifold including flow control orifice.
10. The device in claim 1, wherein elution is performed tangential
to a surface of each filter.
11. A system for fractionation and concentration of particles from
a fluid sample, the system comprising: a reservoir holding a fluid
sample; a fractionation and concentration cartridge including two
or more staged filters; a permeate pressure device in fluid
communication with the cartridge; a concentrating unit including an
actuating integral valving to move sample through the cartridge;
and a fluid dispenser source for collecting concentrated samples
from the cartridge staged filters; wherein the fluid sample is
moved through the concentrating unit, then the concentrated samples
are eluted from the filters and dispensed.
12. The system in claim 11, wherein a flow sensor is in fluid
communication with the sample flow into the cartridge.
13. The system in claim 11, wherein a flow sensor is in fluid
communication with the permeate flow out of the cartridge.
14. A method for rapid fractionation and concentration of particles
from a fluid sample, the method comprising: introducing a sample
into the sample reservoir; initiating a fractionation and
concentration cycle; passing the fluid sample through a series of
filters; eluting a plurality of particles of decreasing particle
size from each filter stage; and extracting a concentrated sample
from each filter stage.
15. The method in claim 14, wherein the eluting further comprises
tangential flushing of a porous surface within the cartridge with
an elution fluid.
16. The method in claim 15, wherein the elution fluid is one or
more of a liquid elution fluid and a wet foam.
17. The method in claim 14, wherein blinding of the filter is
prevented using one or more of high-frequency backpulsing and
oscillating tangential flow.
Description
[0001] This U.S. Patent Application is a continuation of Ser. No.
14/058,193, filed Oct. 18, 2013, now U.S. Pat. No. 10,845,277;
which claims priority to U.S. Provisional Patent Application Ser.
No. 61/715,451, filed Oct. 18, 2012, the content of which is hereby
incorporated by reference herein in its entirety into this
disclosure.
BACKGROUND OF THE SUBJECT DISCLOSURE
Field of the Subject Disclosure
[0003] The subject disclosure relates generally to the field of
sample preparation. More particularly, the subject disclosure
relates to devices, systems and methods for fractionating and
concentrating substances within a fluid sample.
Background of the Subject Disclosure
[0004] The difficulties of detecting and quantifying particles in
air and liquids are well known. Existing systems all begin to fail
as concentration falls away until eventually, with diminished
concentrations of analyte, there is an inability to detect at all.
This poses a significant problem for national security where, for
example, the postal anthrax attacks of 2001 and the subsequent war
on terrorism have revealed shortcomings in the sampling and
detection of biothreats. The medical arts are similarly affected by
the existing limits on detection, as are the environmental
sciences.
[0005] In the fields of biodefense and aerosol research it is
common to collect aerosols into a liquid sample using a wet cyclone
or similar device. The aerosol is collected into an aqueous sample
so that subsequent analysis of biological particles can be
performed using standard techniques that primarily require that the
sample be contained in liquid. These "wet" collectors have many
failings, including: difficulty in maintaining a set fluid volume,
difficulties with buildup of particle matter in the device, and
requirements for storage of the fluid in varying environmental
conditions.
[0006] Dry filters have long been used for collection of aerosols,
as well as for collection of particles from liquids. However, dry
filters fail primarily for the use of identifying biological
particles because detectors generally require a liquid sample and
it is extremely difficult to remove the particles into a liquid.
Methods for removing particles from flat filters are common but are
tedious, inefficient, and require large liquid volumes.
[0007] Concentration of particles from a liquid is traditionally
performed using centrifugation. Centrifugal force is used for the
separation of mixtures according to differences in the density of
the individual components present in the mixture. This force
separates a mixture forming a pellet of relatively dense material
at the bottom of the tube. The remaining solution, referred to as
the supernate or supernatant liquid, may then be carefully decanted
from the tube without disturbing the pellet, or withdrawn using a
Pasteur pipette. The rate of centrifugation is specified by the
acceleration applied to the sample, and is typically measured in
revolutions per minute (RPM) or g-forces. The particle settling
velocity in centrifugation is a function of the particle's size and
shape, centrifugal acceleration, the volume fraction of solids
present, the density difference between the particle and the
liquid, and viscosity of the liquid.
[0008] Problems with the centrifugation technique limit its
applicability. The settling velocity of particles in the micron
size range is quite low. Consequently, centrifugal concentration of
these particles takes several minutes to several hours. The actual
time varies depending on the volume of the sample, the equipment
used, and the skill of the operator.
[0009] Centrifugation techniques are tedious in that they are
normally made up of multiple steps each requiring a high level of
concentration from the operator. Most microbiology laboratories
process large numbers of samples by centrifugation on a daily
basis. The potential for human error is high due to the tedious
nature and automation of these techniques is difficult and costly.
Centrifugation also generally requires powered equipment. Thus,
many situations, such as emergency response, prevent their use.
[0010] Other concentration techniques have been explored and
primarily fall into three technology
groups--microfluidic/electrophoretic based, filtration based, and
capture based. However, each of these techniques has disadvantages
that prevent their use in certain situations.
SUMMARY OF THE SUBJECT DISCLOSURE
[0011] In light of the limitations of conventional techniques, what
is needed is a single device for fractionating and concentrating a
fluid sample into several component concentrations.
[0012] In so doing, the present subject disclosure presents novel,
rapid, efficient one-pass membrane filter based fractionation and
concentration devices, systems and methods that fractionate and
concentrate particles, and especially biological particles
suspended in liquid from a dilute feed suspension ("feed") into
size fractioned and concentrated sample suspensions (retentate),
eliminating the separated fluid (permeate) in a separate stream.
The subject disclosure is particularly useful for the fractionation
and concentration of suspended biological particles, such as
proteins/toxins, viruses, DNA, and bacteria in the size range of
approximately 0.001 micron to 20 microns diameter. Concentration of
these particles is advantageous for detection of target particles
in a dilute suspension, because concentrating them into a small
volume makes them easier to detect. Fractionation is performed in
"cascade" fashion, in order to concentrate particles below the size
cut of each preceding stage remaining in the separated fluid in a
concentrated sample suspension. This process can also be used to
create a "band-pass" concentration for concentration of a
particular target size particle within a narrow range. The device
uses pressure on the feed side, vacuum on the permeate side, and/or
mechanical shear to accelerate the separation process, and may
include an added surfactant to increase efficiency. Integrated
pneumatic, hydraulic, or mechanical valving and a novel vacuum
startup procedure allow for startup of wet membranes while reducing
liquid hold-up volume in the device. The cascade filter stack is
unique in that the sample flow is perpendicular to the surface of a
stack of filters, in series, enclosed in a housing with only a
small open interstitial space between each filter with elution of
the filters performed by a simultaneous wet foam elution performed
parallel, or tangential, to the retentate filter surface through
the small interstitial space. Foam elution is performed
simultaneously one each of the filter stages, so that transmembrane
pressure across each membrane during elution remains essentially
zero or near to it. In this way, flow of elution fluid through the
membranes is eliminated or significantly reduced, so that the
tangential flow velocity and elution efficiency are maximized. The
extraction foam can be prepared from pressurized gas and a
surfactant dissolved in the collection fluid.
[0013] In one exemplary embodiment, the present subject disclosure
is a device for fractionation and concentration of particles from a
fluid sample. The device includes a cartridge containing staged
filters having porous surface in series of decreasing pore size for
capture of particles from a fluid sample; and a permeate pressure
source in fluid communication with the cartridge; wherein the
particles are eluted from the porous surfaces and dispensed in a
reduced fluid volume.
[0014] In another exemplary embodiment, the present subject
disclosure is a system for fractionation and concentration of
particles from a fluid sample. The method includes a reservoir
holding a fluid sample; a fractionation and concentration cartridge
including two or more staged filters; a permeate pressure device in
fluid communication with the cartridge; a concentrating unit
including an actuating integral valving to move sample through the
cartridge; and a fluid dispenser source for collecting concentrated
samples from the cartridge staged filters; wherein the fluid sample
is moved through the concentrating unit, then the concentrated
samples are eluted from the filters and dispensed.
[0015] In yet another exemplary embodiment, the present subject
disclosure is a system for rapid fractionation and concentration of
particles from a fluid sample. The system includes introducing a
sample into the sample reservoir; initiating a fractionation and
concentration cycle; passing the fluid sample through a series of
filters; eluting a plurality of particles of decreasing particle
size from each filter stage; and extracting a concentrated sample
from each filter stage.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1A shows a manifold portion of a five stage fluidics
device, according to an exemplary embodiment of the present subject
disclosure.
[0017] FIG. 1B shows a clamp portion of a five stage fluidics
device, according to an exemplary embodiment of the present subject
disclosure.
[0018] FIG. 1C shows an exploded view of a five stage fluidics
device, according to an exemplary embodiment of the present subject
disclosure.
[0019] FIG. 2 shows an internal fluid volume view of a five stage
fluidics device, according to an exemplary embodiment of the
present subject disclosure.
[0020] FIG. 3 shows an internal fluid volume view of a two stage
fluidics device, according to an exemplary embodiment of the
present subject disclosure.
[0021] FIG. 4 shows an internal fluid volume view of a three stage
fluidics device, according to an exemplary embodiment of the
present subject disclosure.
[0022] FIG. 5 shows a flow chart for fractionation and
concentration, according to an exemplary embodiment of the present
subject disclosure.
[0023] FIG. 6 shows a two cartridge system, according to an
exemplary embodiment of the present subject disclosure.
[0024] FIG. 7A shows a cross sectional view of a two stage filter
stack with integral filter supports, according to an exemplary
embodiment of the present subject disclosure.
[0025] FIG. 7B shows a cross sectional view of a two stage filter
stack with no filter support, according to an exemplary embodiment
of the present subject disclosure.
[0026] FIGS. 8A-8X show a flow chart with detailed steps of a
process for fractionation and concentration, according to an
exemplary embodiment of the present subject disclosure.
DETAILED DESCRIPTION OF THE SUBJECT DISCLOSURE
[0027] The present subject disclosure relates generally to the
fields of bioterrorism security, medicine, and environmental
science. Rapid, reliable detection of airborne biothreats is a
significant need for the protection of civilians and military
personal from pandemic outbreaks and bioterrorist events. Best in
class biothreat detection systems use aerosol collectors to capture
particles into a liquid volume in the range of 2 to 12 mL. Samples
are then processed using a number of sample preparation techniques
and analyzed by rapid microbiological methods, including real-time
quantitative polymerase chain reaction (qPCR) and ultra-high
throughput sequencing (UHTS) and/or gold-standard culture based
methods. While the state of the art for rapid detectors,
collectors, and identifiers has advanced dramatically in recent
years, advancement of sample preparation techniques has lagged
significantly and considerable improvements are needed in these
techniques.
[0028] Detect/collect/identify systems for airborne biothreats must
operate correctly in all types of indoor and outdoor environments.
Urban, industrial, and rural outdoor environments as well indoor
environments range from very low to very high particle
concentrations. Detection of threats in these varied environments
often hinges on the ability of the system to capture and identify
rare threat particles in what can be a highly varied, complex
mixture of organic and inorganic debris particles, innocuous
microbes, pollen, fungal spores, and mammalian cells.
[0029] Better automated sample preparation techniques are needed so
problems currently associated with detection of rare particles in
complex environmental samples can be overcome. Inhibition of
identification techniques due to environmental debris is a common
problem with these systems due to the varied, high-level complex
mixtures of particle and chemical inhibitors. UHTS, qPCR, and other
rapid detection techniques can also fail due to high levels of
background clutter. Breakdown of bioinformatic systems used for
UHTS data analysis due to high background clutter levels is one of
the biggest hurdles that must be overcome before cutting-edge
sequencing can be adapted to autonomous biothreat detection
applications. There is also a significant requirement to be able to
differentiate between target agents coming from whole, viable cells
and those present as free DNA or free proteins. The inability to
rapidly determine if the target particle is a whole viable cell or
is only present as free DNA or protein signature, as is the norm in
today's biothreat detection systems, does not allow organizations
to differentiate between what may be an actual terrorist event from
potentially catastrophic false alarms associated with hoaxes or
natural events.
[0030] Aerosol samples and other samples of importance (e.g.,
surface, liquid, clinical, food, etc.), often contain a significant
amount and wide range of non-target debris including organic and
inorganic matter and biological materials. As described above,
these non-target materials can significantly affect the performance
of sample preparation and agent identification techniques with a
common side effect of inhibition. Conventional sample preparation
techniques exist for removing these inhibitors, but they are slow
and perform best when volumes of only a few hundred microliters are
processed--demonstrating the mismatch between collected sample size
and the volume that can be processed and analyzed by available
technologies. This mismatch raises the true system detection limit
to levels significantly higher than the desired detection limit and
creates a significant likelihood of false negative results when, as
would typically be the case, only trace levels of signature are
present.
[0031] A wide range of existing, and developing, rapid analysis
platforms are potentially useful technologies for detection and
identification needs. Detection and identification may key on whole
organisms, nucleic acids, or proteins. Culture based analysis,
antibiotic susceptibility testing, and functional assays all
require live organism samples. Common nucleic acid techniques
include qPCR, UHTS, and hybridization arrays. ELISA and other
immunoassay techniques, mass spectrometry, chromatography
techniques, and other techniques may be used for protein analysis.
There are significant reasons in some cases to choose one of these
techniques over the other or in some cases to analyze with more
than one technique. Additionally some techniques lend themselves to
use in autonomous detection platforms and some are used only in
laboratory settings. Further, it is difficult to determine what
techniques may receive precedence in the near future as costs fall
or new improved methods are developed. This difficulty in
determining what detection and identification system may be used
warrants the need for a plug-and-play type of sample preparation
system that is capable of delivering the needed sample fractions in
a concentrated form for each potential type of analysis.
[0032] Robust, fast, and sensitive detection systems are needed,
but currently most systems fail to meet these needs due to
deficiencies in sample preparation. The sample preparation system
must be capable of autonomous operation for a month or more without
maintenance. The same environmental particles and inhibitors that
commonly cause issues with the identifier can also lead to failure
of the sample preparation system, especially after repeated use
over extended periods of time. The time required for the sample
preparation methods used for these complex samples is a large
portion of the total time needed for identification and, even so,
the methods are only capable of processing a very small portion of
the available sample.
[0033] The present subject disclosure presents a novel technique of
fractionating multiple components simultaneously. It may be used in
numerous fields, including, but not limited to, bioterrorism
detection. For example, exemplary and specific fields of use
include, but are not limited to: [0034] 1. Aerosol sampling for
bioterrorism threat agents [0035] a. Where the sample results in a
liquid sample for analysis [0036] b. Where the sample can contain
target agent(s) that are thought to be a substantial threat to the
health of humans [0037] i. Where a list of the potential threat
(target) agent(s) can be taken from the U.S. Food and Drug
Administration's Centers for Disease Control and Prevention (CDC)
Select Agents A, B, or C list (See List 1, below) [0038] c. Where
the sample can contain target agent(s) that are thought to be a
threat to the health of humans, animals or plants, causing societal
disruption and economic harm [0039] i. Where a list of the
potential threat (target) agents can be taken from the CDC agent
list (http://www.bt.cdc.gov/agent/agentlist.asp), or List 2, below
[0040] d. Where the resulting sample can contain test particles,
target agent(s) or surrogate(s) in a concentration too small for
detection by the chosen method [0041] i. Where concentration of the
sample into a smaller volume can result in detection of the threat
agent(s) of interest by one or a combination of the following
methods: [0042] 1. Where detection of the threat agent(s) is
performed by polymerase chain reaction (PCR) or PCR-like methods
[0043] 2. Where detection of the threat agent(s) of interest is
performed by immunoassay methods [0044] 3. Where detection of the
threat agent(s) of interest is performed by ultraviolet light
fluorescence methods [0045] ii. Or where concentration and analysis
resulting in a non-detect result can provide assurance that if the
target agent is present, it is present in such a low quantity that
the resulting risk to the affected population is minimal [0046]
iii. Where separation of the sample into desirable size fractions
can concentrate the target particles into separate but equally
concentrated size fractions for analysis by different detection
methods listed in 1.c.i. above, such as: [0047] 1. Separating and
concentrating particles larger than 0.2 microns to separate and
concentrate bacteria [0048] iv. Where a small size range or
"band-pass" can be separated out and concentrated for interrogation
for a particular threat agent or surrogate, such as: [0049] 1.
separating and concentrating particles from 0.2 microns diameter to
2 microns diameter to separate bacterial spores and concentrate
them separately from smaller and larger particles present in the
initial sample [0050] 2. separating and concentrating particles
from 0.005 microns to 0.2 microns diameter to separate most viruses
and concentrate them separately from smaller and larger particles
present in the initial sample (examples include viral equine
encephalitis, or VEE; 0.06 microns diameter). [0051] 3. separating
and concentrating particles from 0.001 microns (approximately 5
kiloDaltons) to 0.01 microns (approximately 100 kiloDaltons) to
separate toxins and proteins and concentrate them separately from
smaller and larger particles present in the initial sample [0052]
2. The above types of sampling and analysis are performed for the
fields of homeland security, corporate security, and military force
protection: [0053] a. Automated sampling and analysis systems such
as those developed for government programs Portal Shield, Joint
Programs Biological Detection System (JPBDS), US Postal Service
Biological Detection System (BDS), and systems under development,
such as the Biological Aerosol Networked Detection (BAND) system
and Rapid Aerosol Biological Identification System (RABIS) [0054]
b. Manual systems such as bioaerosol collection using air/liquid
impingers, including the All Glass Impinger (AGI-30, Ace Glass,
Inc., Vineland, N.J.), Greenburg-Smith impingers, and SKC
Biosamplers provide samples that are in the 20-100 mL size range,
and can be concentrated down to the 4-400 uL volume range using the
InnovaPrep device and process described here [0055] c. Samples
resulting from manual swabbing of surfaces onto wetted swabs, pads,
or pieces of filter material are often taken for bioterrorism
security monitoring and are typically extracted into a volume of
liquid resulting in a 2 to 20 mL volume initial sample. Samples
like these can be quickly concentrated to much smaller volumes in
the range of 4-400 uL using the InnovaPrep [0056] 3. Water sampling
for bioterrorism threat agents [0057] a. Where the sample can
contain target agent(s) that are thought to be a substantial threat
to the health of humans by ingestion or contact [0058] i. Where a
list of the potential threat (target) agent(s) can be taken from
the U.S. Food and Drug Administration's Centers for Disease Control
and Prevention (CDC) Select Agents A, B, or C list (See List 1,
below) [0059] b. Where the sample can contain target agent(s) that
are thought to be a threat to the health of humans, animals or
plants, causing societal disruption and economic harm [0060] i.
Where a list of the potential threat (target) agents can be taken
from the CDC agent list
(http://www.bt.cdc.gov/agent/agentlist.asp), or List 2, below
[0061] c. Where the resulting sample can contain test particles,
target agent(s) or surrogate(s) in a concentration too small for
detection by the chosen method [0062] i. Where concentration of the
sample into a smaller volume can result in detection of the threat
agent(s) of interest by one or a combination of the following
methods: [0063] 1. Where detection of the threat agent(s) is
performed by polymerase chain reaction (PCR) or PCR-like methods
[0064] 2. Where detection of the threat agent(s) of interest is
performed by immunoassay methods [0065] 3. Where detection of the
threat agent(s) of interest is performed by ultraviolet light
fluorescence methods [0066] ii. Or where concentration and analysis
resulting in a non-detect result can provide assurance that if the
target agent is present, it is present in such a low quantity that
the resulting risk to the affected population is minimal [0067]
iii. Where separation of the sample into desirable size fractions
can concentrate the target particles into separate but equally
concentrated size fractions for analysis by different detection
methods listed in 1.c.i. above, such as: [0068] 1. Separating and
concentrating particles larger than 0.2 microns to separate and
concentrate bacteria [0069] iv. Where a small size range or
"band-pass" can be separated out and concentrated for interrogation
for a particular threat agent or surrogate, such as: [0070] 1.
separating and concentrating particles from 0.2 microns diameter to
2 microns diameter to separate bacterial spores and concentrate
them separately from smaller and larger particles present in the
initial sample [0071] 2. separating and concentrating particles
from 0.005 microns to 0.2 microns diameter to separate most viruses
and concentrate them separately from smaller and larger particles
present in the initial sample (examples include viral equine
encephalitis, or VEE; 0.06 microns diameter). [0072] 3. separating
and concentrating particles from 0.001 microns (approximately 5
kiloDaltons) to 0.01 microns (approximately 100 kiloDaltons) to
separate toxins and proteins and concentrate them separately from
smaller and larger particles present in the initial sample [0073]
4. The above types of sampling and analysis are performed for the
fields of homeland security, corporate security, and military force
protection: [0074] a. Water samples taken from water sources used
to produce potable water for consumption by the public or
government use [0075] b. Water samples taken to determine a source
of production of bioterrorism agents [0076] c. Water samples taken
to determine whether biological decontamination has been effective
[0077] 5. Agricultural samples for bioterrorism threat agents
[0078] a. Where the sample can contain target agent(s) that are
thought to be a substantial threat to the health of plants or
animals, or indirectly to humans after ingestion of contaminated
agricultural products [0079] b. Where the sample is liquid or can
be extracted into a liquid for analysis [0080] i. Where a list of
the potential threat (target) agent(s) can be taken from the U.S.
Food and Drug Administration's Centers for Disease Control and
Prevention (CDC) Select Agents A, B, or C list (See List 1, below)
[0081] c. Where the sample can contain target agent(s) that are
thought to be a threat to the health of humans, animals or plants,
causing societal disruption and economic harm [0082] i. Where a
list of the potential threat (target) agents can be taken from the
CDC agent list (http://www.bt.cdc.gov/agent/agentlist.asp), or List
2, below [0083] d. Where the resulting sample can contain test
particles, target agent(s) or surrogate(s) in a concentration too
small for detection by the chosen method [0084] i. Where
concentration of the sample into a smaller volume can result in
detection of the threat agent(s) of interest by one or a
combination of the following methods: [0085] 1. Where detection of
the threat agent(s) is performed by polymerase chain reaction (PCR)
or PCR-like methods [0086] 2. Where detection of the threat
agent(s) of interest is performed by immunoassay methods [0087] 3.
Where detection of the threat agent(s) of interest is performed by
ultraviolet light fluorescence methods [0088] ii. Or where
concentration and analysis resulting in a non-detect result can
provide assurance that if the target agent is present, it is
present in such a low quantity that the resulting risk to the
affected population is minimal [0089] iii. Where separation of the
sample into desirable size fractions can concentrate the target
particles into separate but equally concentrated size fractions for
analysis by different detection methods listed in 1.c.i. above,
such as: [0090] 1. Separating and concentrating particles larger
than 0.2 microns to separate and concentrate bacteria [0091] iv.
Where a small size range or "band-pass" can be separated out and
concentrated for interrogation for a particular threat agent or
surrogate, such as: [0092] 1. separating and concentrating
particles from 0.2 microns diameter to 2 microns diameter to
separate bacterial spores and concentrate them separately from
smaller and larger particles present in the initial sample [0093]
2. separating and concentrating particles from 0.005 microns to 0.2
microns diameter to separate most viruses and concentrate them
separately from smaller and larger particles present in the initial
sample (examples include viral equine encephalitis, or VEE; 0.06
microns diameter). [0094] 3. separating and concentrating particles
from 0.001 microns (approximately 5 kiloDaltons) to 0.01 microns
(approximately 100 kiloDaltons) to separate toxins and proteins and
concentrate them separately from smaller and larger particles
present in the initial sample [0095] v. Where exclusion of
interferent particles such as diesel soot is desirable to improve
the performance of the analysis method [minimization of
interference or improvement of "contrast" may be desirable for all
fields] [0096] 6. The above types of sampling and analysis are
performed for the fields of homeland security, corporate security,
and military force protection: [0097] a. Where foodstuffs such as
milk is monitored for toxin contamination such as by ricin [0098]
b. Where meatpacking plants are monitored for biological
contamination by E. coli, Listeria spp. Such monitoring is also
conducted for quality assurance, such as hazard assessment and
critical control point (HACCP) programs [0099] c. For bottled water
production
[0100] The present subject disclosure may be used to assist in
identifying agents from the following lists:
[0101] List 1: CDC Category A and B Bioterrorism Agents List
Category A (Definition Below)
[0102] Anthrax (Bacillus anthracis) Botulism (Clostridium botulinum
toxin) Plague (Yersinia pestis) Smallpox (variola major) Tularemia
(Francisella tularensis) Viral hemorrhagic fevers (filoviruses
[e.g., Ebola, Marburg] and arenaviruses [e.g., Lassa, Machupo])
Category B (Definition Below)
[0103] Brucellosis (Brucella species) Epsilon toxin of Clostridium
perfringens Food safety threats (e.g., Salmonella species,
Escherichia coli O157:H7, Shigella) Glanders (Burkholderia mallei)
Melioidosis (Burkholderia pseudomallei) Psittacosis (Chlamydia
psittaci) Q fever (Coxiella burnetii) Ricin toxin from Ricinus
communis (castor beans) Staphylococcal enterotoxin B Typhus fever
(Rickettsia prowazekii) Viral encephalitis (alphaviruses [e.g.,
Venezuelan equine encephalitis, eastern equine encephalitis,
western equine encephalitis]) Water safety threats (e.g., Vibrio
cholerae, Cryptosporidium parvum)
[0104] List 2: Secondary Potential Biological Threat Agents
Viri/Prions
[0105] Flaviviruses (Yellow fever virus, West Nile virus, Dengue,
Japanese Encephalitis,
TBE, etc.)
Hep A, B, C
Prions (CJD, BSE, CWD)
Alphaviruses (VEE, EEE, WEE)
[0106] Nipah virus Rabies virus Rhinovirus (could be modified?)
Polioviruses
Hantaviruses
Filoviruses (Ebola, Marburg, Lassa)
Bacilli
[0107] Mycobacterium tuberculosis, drug resistant Mycobacteria
other than TB, like C. leprae Streptococcus pneumoniae S. pyogenes
S. aureus Clostridium tetani C. difficile Bacillus cereus Coxiella
brunette (Q fever) Francisella tularensis Borrelia recurrentis
Rickettsia rickettsii R. prowazekii Shigella sonnei Bartonella
henselae Yersinia enterolitica Y. pseudotuberculosis Neisseria
meningitidis Legionella pneumophila Burkholderia pseudomallei
Pasteurella multocida
Other Pathogenic Microorganisms
[0108] Cryptosporidium parvum Histoplasma capsulatum Cryptococcus
neoformans Aspergillus niger
Pathogenic Fungi
Acremomium spp.
[0109] Alternaria alternate Apophysomyces elegans Aspergillus
terreus
Bipolaris spp.
[0110] Bipolaris spicifera Blastoschizomyces capitatus Candida
krusei Candida lusitaniae Cladophialophora bantiana Cunnihamella
berholletiae Curvularia lunata Exserohilum rostratum Fusarium
moniliforme Fusarium solani Hansenula anomala Lasiodilodia
theobromae Malassezia furfur Paecilomyces lilacinus Paecilomyces
bariotii Penicillium marneffei Phialemonium curvatum Philophora
parasitica P. richardsiae
Ramichloridium spp.
[0111] Rhizomucor pusillus Rhizopus rhizopodiformus Rhodotorula
rubra Saccharomyces cerevisiae Scedosporium prolificans
Trichosporon beigelii (T. asahii) Wangiella dermatitidis
[0112] The present subject disclosure may be used to assist in
identifying various agents of varying sizes:
Definition of Category a Diseases/Agents
[0113] The U.S. public health system and primary healthcare
providers must be prepared to address various biological agents,
including pathogens that are rarely seen in the United States.
High-priority agents include organisms that pose a risk to national
security because they [0114] can be easily disseminated or
transmitted from person to person; [0115] result in high mortality
rates and have the potential for major public health impact; [0116]
might cause public panic and social disruption; and [0117] require
special action for public health preparedness.
Definition of Category B Diseases/Agents
[0118] Second highest priority agents include those that [0119] are
moderately easy to disseminate; [0120] result in moderate morbidity
rates and low mortality rates; and [0121] require specific
enhancements of CDC's diagnostic capacity and enhanced disease
surveillance.
Definition of Category C Diseases/Agents
[0122] Third highest priority agents include emerging pathogens
that could be engineered for mass dissemination in the future
because of [0123] availability; [0124] ease of production and
dissemination; and [0125] potential for high morbidity and
mortality rates and major health impact
Physical Sizes of Some Agents and Surrogates:
[0126] Target:
TABLE-US-00001 Bacillus thuringiensis endospore - approximately 1
.mu.m Bacillus anthracis endospore - approximately 1 .mu.m Yersinia
pestis - Gram negative rod-ovoid 0.5-0.8 .mu.m in width and 1- 3
.mu.m in length Yersinia rohdei - approximately 1 .mu.m Venezuelan
Equine Encephalitis - 70 nm (0.07 .mu.m) Gamma-killed MS2 - 2 mD or
about 25 nm (0.025 .mu.m) (but will pass through a 300 kD pore size
but is retained by a 100 kD pore size Wick and McCubbin - ECBC)
Ovalbumin - 45 kD or 6 nm (0.006 .mu.m) Botulinum Toxoid A - 150 to
900 kD or 10 nm to 70 nm (0.01 .mu.m to 0.07 .mu.m)(Normally
published as 150 kD however some publications state that toxoid A
can be released as complexes comprised of the 150 kD toxin protein
along with associated non-toxin proteins and can therefore be
released in 900 kD, 500 kD, and 300 kD forms. DNA - 1000 Bp or 600
kD up to 15,000 Bp or 9 mD
[0127] Specific fields of use in the medical field include, but are
not limited to: [0128] 1. The above types of sampling and analysis
are performed for the fields of medical research and diagnostics:
[0129] a. In cancer research where very low concentrations of
experimental drugs in body fluids or urine are the targets of
analysis [0130] b. In allergy diagnosis where low quantities of
specific antigens are the targets of analysis in body fluids [0131]
c. In health effects research regarding the determination of health
effects known to be caused by various materials in inhaled
particulate matter with aerodynamic diameter below 2.5 microns (PM
2.5). this area overlaps with environmental studies (see below).
[0132] d. In forensic medicine where low concentrations of toxins
or venoms are the targets of analysis in body fluids [0133] e. In
operating rooms [surface extraction and air monitoring, add] [0134]
f. In pharmaceutical manufacturing where the biological aerosol
particulate matter concentration is regulated by the US Food and
Drug Administration
[0135] Specific fields of use in the environmental studies field
include, but are not limited to:
[0136] [similar to outline above, modified to fit the environmental
applications] [0137] 2. The above types of sampling and analysis
are performed for the field of environmental study: [0138] a. In
health effects research regarding the determination of health
effects known to be caused by various materials in inhaled
particulate matter with aerodynamic diameter below 2.5 microns (PM
2.5) [0139] b. High altitude aerosol research where low quantities
of particulate are collected and must be concentrated for study
[0140] c. In cleanrooms where very low aerosol concentrations of
aerosol particles are collected for monitoring aimed at source
control [0141] d. For separation of populations of particles
collected at different heights above the ground (profiling
studies)
[0142] The present subject disclosure has been developed as a
unique membrane filter based fractionation and concentration system
that is capable of separating particles by size and concentrating
those particles into small (<100 .mu.L) sample volumes. A novel
approach was developed in which the membrane filters are stacked,
in order of decreasing pore size, inside a single cartridge with a
small interstitial space, or in some cases a solid filter support
and further reduced interstitial space, between each membrane
filter. Sample flow is introduced perpendicular to the first filter
surface and is pushed or pulled, in series, directly through each
of the membrane in the cartridge. Because the cartridge can be
designed for reuse, and because wet hydrophilic membrane filters
will not allow air to flow through at pressures below the bubble
point, a novel vacuum startup method is used to allow air to be
removed from the interstitial space and other internal volume, so
that the sample process can be initiated. A series of channels and
associated valves, integral to the cartridge, are used to link each
stage back to a pump to allow for negative pressure to be pulled on
the system.
[0143] After negative pressure has been pulled on the system, the
sample flow is introduced as described above. The entire sample is
flowed through the cartridge, until air reaches the first membrane
filter and the system locks up. The vacuum startup valves are then
actuated one by one to allow the remaining fluid to be pushed
through the remaining membrane filters. When then entire sample
volume has been processed then the cartridge inlet and outlet
valves are closed and a retentate valve is opened on each stage. A
wet carbonated foam is then introduced into one end of the
cartridge, which subsequently travels the length of the cartridge,
tangential to the retentate surface of each membrane. Finally the
foam is dispensed out of the retentate port into a separate sample
container for each membrane filter. The foam then breaks down into
a liquid leaving a small concentrate fraction associated with each
membrane filter stage.
[0144] The subject disclosure of the present application, which
describes liquid-to-liquid fractionation and concentration devices,
systems and methods, provides a novel means of rapidly and
efficiently separating and then concentrating biological samples.
Significant advantages are offered over current methods including,
but not limited to: improved separation efficiency, improved
concentration efficiency, shorter process times, automation, and
integration into automated systems. Like centrifugation,
filtration, and the other conventional methods, this present
technique concentrates the collected sample prior to analysis, but
with many further advantages, including but not limited to: 1) the
liquid volume of the sample is quickly reduced. Unlike
centrifugation, which typically takes 10 to 30 minutes to
concentrate micron-sized particles, this process can be
accomplished in 5 to 60 seconds for a 10 mL initial volume. Unlike
conventional hollow fiber filter concentration, in which the
initial sample is recycled many times through the filter taking
from several minutes to hours in order to concentrate a particle
such as a protein or enzyme into a volume of several milliliters,
the sample is passed straight through in one pass. This results in
a much smaller volume of liquid on the order of 100 to 400
microliters, or passed straight through in dead-end fashion and
then extracted in a volume of liquid or foam in the range of 4 to
400 microliters. Unlike typical single-pass flat filtration, the
sample remains in liquid form for transport and analysis. The
detection limit for the target agent is lowered, with respect to
the media originally sampled. 2) The final sample volume is reduced
much further than in previously known methods, while kept in liquid
form, allowing detection in devices such as multi-well plate
readers that utilize small input samples. 3) The reduced-size
samples can be more efficiently stored and transported by
microfluidic handling methods. 4) The device can be constructed to
separate particles in one pass into different size fractions for
analysis for certain agents. For example, cells and spores can be
concentrated separately from viruses and biological toxins.
Further, the size range that is concentrated can be narrow, or
"band-pass" to concentrate a small size range fraction from a
complex matrix, such as an environmental sample 5) The device can
be used to reduce the onboard fluid storage capacity of aerosol
samplers, by recycling the cleaned liquid back to the collection
cycle after the sampled particles are removed into a small volume
for analysis. 6) This device is much more readily adapted to
automated systems than other technologies including centrifugation,
flat filtration, and other methods. The flow-through nature of the
device allows for straightforward configuration into an automated
detection system. 7) This device is significantly more robust in
nature than new microfluidic concentration systems such as
dielectrophoresis concentration systems. Dielectrophoresis systems
developed by Sandia have internal flow paths of small diameters
that can create significant clogging during processing of fluids
with high particle concentrations. Commercially available hollow
fiber filters, while possessing pores of up to a maximum of
approximately 0.5 .mu.m diameter, will take significantly longer to
clog, due to the high number of pores and the tangential flow
cleaning with the preferred surfactant foam. 8) The InnovaPrep
system is much smaller than any commercially available liquid to
liquid concentrator. Necessary components can be arranged in such a
way as to take advantage of any empty space in the system being
integrated. 9) The device it made almost entirely of low cost,
readily available components. This significantly lowers the cost of
integration and makes it more practical than other methods
concentration.
[0145] An exemplary embodiment of a device according to the present
subject disclosure is presented in FIGS. 1A-1C. In these figures,
an exemplary device 100, which can be used for fractionation and
concentration of components within a fluid, is presented. The
device 100 includes a manifold portion 101, with a manifold
mounting flange 111 used to connect or secure the device, and a
clamp plate 102 which together serve as end pieces to the device
100, and the fluidic stack 108 are held within those end pieces.
Clamping bolts 103 maintain a sealed condition for the device 100
when it is operated. Alignment pins 104 serve to maintain the
structural integrity of the device and provide an easier method to
put all components together. Fluid connections 105 are the ports
where fluid is introduced into the device 100. Pneumatic control
line 106 regulates the pressure within the device 100.
[0146] FIG. 1C shows an exploded view of a laminated multi-stage
concentration cell device 100 with bolts 103 and alignment pins 104
removed for sake of clarity. The fluidic stack 108 comprises of
numerous layers of hard plastic layers 121 with holes cut out in
specific shapes and geometries and fluid paths etched in the
surface of, and gaskets 122 enclosing filter portions 123. The
device 100 is constructed by compressing alternating layers of
plastic 121 and filter media 123 between two fluidic blocks 122
which allow the cell to interface with the rest of the fluidic
system. This type of cartridge may be constructed using the method
shown or may be constructed using bonding and construction
techniques that are commonly used in microfluidic device
construction. An exemplary cartridge is shown having 5 filters. In
this case, the 5 membrane filters could be made up with filter
types, or similar filter types to that shown below. [0147] Filter
1-6 .mu.m track-etched polycarbonate membrane filter for large
particle removal [0148] Filter 2--Affinity based filter for removal
of humics [0149] Filter 3--0.4 .mu.m track-etched polycarbonate
membrane filter for bacteria capture [0150] Filter 4--0.02 .mu.m
block copolymer membrane filter for virus and nucleic acid capture
[0151] Filter 5--10 kD block copolymer membrane filter for protein
capture In this way the system would produce fractions of target
particles containing the following particle types, with reduced
numbers or concentrations of interfering particles or humics.
[0152] Whole Bacteria [0153] Viruses and free nucleic acids [0154]
Proteins
[0155] FIG. 7A shows a cross sectional view of a two stage filter
stack with integral filter supports. This cell is constructed by
laminating several layers of different material together to create
fluid channels. The components are (1) ridged plastic substrate in
which fluid paths are etched, (2) soft plastic substrate which
functions as a gasket creating a gas and liquid tight seal between
layers, (3) filtration media, (4) filter support ridges, and (5)
fluid paths connecting one filter stage to the next such that the
permeate of the first stage filter becomes the sample of the second
stage filter.
[0156] FIG. 7B shows a cross sectional view of a five stage filter
stack. This cell is also constructed by laminating layers of
material together; the filter media is arranged such that the
sample travels through all five filter stages in a single step,
starting with the largest pore diameter filter and ending with the
smallest. The components are (6) the sample inlet port, (7) soft or
ridged substrate sealing the layers together and creating fluid
channels, (8) filtration media, (9) the permeate port. (10) shows
the direction of the sample flow.
[0157] It should be noted that although the exemplary embodiment
shown in FIGS. 1A-1C includes five layers of filters, any number is
possible, and the technique to make and use the device will be
similar, and understood by one having ordinary skill in the art
when considering the present disclosure.
[0158] Once the device 100 is properly aligned with alignment pins
104 and securely fastened with bolts 103, a fluidic internal volume
200 is created with numerous chambers, passageways and connections.
Such internal fluid volume 200 is shown in FIG. 2. It should be
noted that this internal fluidic volume is created as a result of
the laser cut passageways of the various hard plastic layers 121,
gaskets 122, and filters 123 used in the fluidic stack 108.
[0159] Internal fluidic volume 200 shows various paths for the
fluidic stack 108 assembly for a five stage concentrator. 202, 204
and 206 are pneumatic control lines, and used to control filter
stage 1 bypass valve (humic acid removal) 202, Filter stage 2
bypass valve (prefilter) 204, and decontamination isolation valves
206.
[0160] Various fluid lines include the decontamination fluid outlet
port 208, the filter stage 3 retentate port (concentration stage 1)
210, the filter stage 1 retentate port (Humic acid removal) 212,
the filter stage 4 retentate port (concentration stage 2) 214, the
filter stage 2 retentate port (Prefilter) 216, and the filter stage
5 retentate port (concentration stage 3) 218.
[0161] Further pneumatic control lines include the filter stage 5
bypass valve (Concentration stage 3) 220, filter stage 4 bypass
valve (concentration stage 2) 222, filter stage 3 bypass valve
(concentration stage 1) 224, and the master filter isolation valve
226.
[0162] Further fluid lines include the gas flush port 228, the foam
injection port 230, the sample inlet port 232, the sample outlet
port (permeate) 234, and the decontamination fluid inlet port 236.
Part of the pneumatic control line is the feed/permeate isolation
valve 238. Finally, the various filter stages include filter stage
1 250, filter stage 2 252, filter stage 3 254, filter stage 4 256,
and filter stage 5 258.
[0163] All of the components and internal fluid channels for the
five stage fluidic stack shown in FIG. 2 work together in the
manner as described in further detail below. It is noted that the
five stage fluidic stack shown in FIG. 2 is merely exemplary, and
that the present disclosure is not limited to such an exemplary
embodiment. For example, a two stage fluidic internal volume is
shown in FIG. 3 and a three stage fluidic internal volume is shown
in FIG. 4. Other numbers are also possible and within the purview
of one having ordinary skill in the art.
[0164] For sake of completeness, the components of the two stage
fluidics internal volume 300 include: [0165] 301 Microvalve control
pneumatic control diaphragm (7 shown) [0166] 302 Micro fluidic
valve (7 shown) [0167] 303 (pneumatic control line) Filter stage 1
bypass valve [0168] 304 (Fluid line) Sample outlet (Permeate)
[0169] 305 (Fluid line) Sample inlet (Feed) [0170] 306 (Fluid line)
Filter stage 1 retentate [0171] 307 (Fluid line) Filter stage 2
retentate [0172] 308 (pneumatic control line) Master filter
isolation valve [0173] 309 (pneumatic control line) Filter stage 2
bypass valve [0174] 310 (Fluid line) Foam injection port [0175] 311
Filter stage 1 [0176] 312 Filter stage 2
[0177] For sake of completeness, the components of the three stage
fluidics internal volume 400 include: [0178] 401 Stage 1 foam inlet
[0179] 402 Stage 1 foam microvalve [0180] 403 Stage 2 foam inlet
[0181] 404 Stage 2 foam microvalve [0182] 405 Stage 3 foam inlet
[0183] 406 Stage 3 foam microvalve [0184] 407 Filter stage 1 bypass
valve [0185] 408 Filter stage 2 bypass valve [0186] 409 Sample
inlet (feed) [0187] 410 Stage 1 retentate outlet [0188] 411 Stage 1
retentate valve [0189] 412 Stage 2 retentate outlet [0190] 413
Stage 2 retentate valve [0191] 414 Stage 3 retentate outlet [0192]
415 Stage 3 retentate valve [0193] 416 Filter stage 1 [0194] 417
Filter stage 2 [0195] 418 Filter stage 3
[0196] A process flow diagram for an exemplary system according to
the present subject disclosure is presented in FIG. 5. The orange
boxes 522, 525, 542, 545, 547, 551 contain the final six
concentrated fractions that will be recovered from the input sample
shown in the red box 501. Two fractionation/concentration fluidics
cartridges 510 and 540 will be used to produce the six fractions
522, 525, 542, 545, 547, 551. Cartridge A 510 will separate the
input sample into fractions containing whole cells 514, free
nucleic acids 522, and free proteins 525. A portion of the whole
cell fraction 514 from Cartridge A 510 will be lysed and then
Cartridge B 540 will be used to separate the whole cell lysate 543
into fractions containing cell debris 545, nucleic acids 547, and
proteins 551. Cartridge A 510 separations will be performed with
stages A.1 511, A.2 513, A.3 515, A.4 521, and A.5 524. Cartridge B
540 will house stages B.1 544, B.2 548, and B.3 550. Prior to
Cartridge A 510, a novel, replenishable media column loaded with
Polyvinylpolypyrrolidone (PVPP) media will be used to remove humic
substances while allowing target materials to pass. Stage A.1 511
will use a large pore membrane to remove environmental debris and
inhibitors 530, including large particulate matter, from the input
sample. Stage A.2 513 will be a novel, replenishable
Polyvinylpolypyrrolidone (PVPP) Sol-gel membrane used to remove
humic substances 531 while allowing target materials to pass. Stage
A.3 515 is used to capture whole viable organisms. A portion of
this fraction is then archived for later analysis and a portion is
lysed for rapid detection. The permeate fluid from this stage will
contain free solution nucleic acids and free solution proteins
which are subsequently separated into a nucleic acid fraction and a
protein fraction with Stages A.4 521 and A.5 524, respectively. The
lysed fraction of whole viable cells, to be used for rapid
detection, is separated into three fractions containing cellular
debris, nucleic acids and proteins with Stages B.1 544, B.2 548 and
B3 550, respectively. The various permeates 512, 514, 516, 523, 549
are shown to indicate the remaining substances of the process.
Permeate waste 526 and 552 indicate the end result of the processes
of cartridge A 510 and cartridge B 540, respectively.
[0197] FIG. 6, in conjunction with FIG. 5, provides a flow
schematic of a layout of an exemplary version of a two cartridge
system 600. FIGS. 5 and 6 should be considered jointly for the
proceeding discussion. During operation, a sample with a volume of
1 mL to 50 mL is fed into the sample input reservoir. A processing
cycle is then initiated. The first step is preparation of the
cartridges 601, 603 for processing using a novel vacuum startup
method. Because the membranes 606 are hydrophilic in nature and are
wet for every sample processed after the first, startup requires
that air be evacuated from the system 600 so that the liquid
samples can be brought into contact with the membranes. It should
be noted that only one membrane 606 is pointed out in the figure
for sake of clarity, but multiple membranes are shown. Pulling
negative pressure on each stage within the cartridges 601, 603
performs this action. Pneumatic valves within the cartridges 601,
603 are activated to allow a vacuum to be applied through a
three-way valve 602, 604 at the top of each cartridge,
respectively. When a sufficient vacuum has been achieved, the
valves are closed so that negative pressure is captured within the
cartridge. The entire vacuum startup process is anticipated to take
less than 20 seconds to perform.
[0198] When the vacuum startup is complete the sample is processed
through a PVPP column for humic removal followed by Cartridge A
520, 601. Fluid then flows through all four stages of Cartridge A
520, 601 in a single pass. Because the interstitial space between
membranes 606 is small (less than 300 .mu.L) and because the
membranes are arranged in series the total hold-up volume in
Cartridge A 520, 601 will be less than 1.5 mL with a processing
rate that is limited primarily only by the slowest membrane in the
cartridge. Total time to process a 10 mL sample through the humic
removal column and Cartridge A 510, 601 is approximately 10
minutes. When the all of the liquid sample passes through the Stage
A.1 511 membrane the system will lock up since air will not pass
through a wet hydrophilic membrane. A Liquid Flow Switch is then
used to determine when the system has locked up and air pressure is
applied to the next stage so that liquid can be pushed through the
next membrane filter. This process is continued until all the
liquid has been evacuated from the system.
[0199] When the entire sample has been processed each Stage is
extracted simultaneously. By performing the extraction process
simultaneously, pressures across each membrane are balanced and
flow through the membranes does not occur since the pressure is
equal on both sides. This process provides for the best possible
concentration efficiencies with the smallest resulting extraction
volume. The extraction process takes place by opening and closing a
single extraction fluid valve connected, through internal cartridge
fluidics, to each stage. The valve is opened for a short period of
time (15 to 50 msec) to allow extraction fluid to be dispensed
rapidly into the interstitial space between each membrane. Once
dispensed the extraction fluid quickly forms wet, viscous foam that
travels the length of the membrane and is dispensed into separate
capture reservoirs for each stage.
[0200] Concentrates released from Cartridge A 510 will include
fractions containing environmental waste debris for disposal, whole
cells, free nucleic acids, and free proteins. The whole cell
concentrate from Cartridge A 510 will be split into an archived
sample and a sample available for secondary processing. The sample
available for secondary processing is then processed using a
flow-though mechanical cell lysis system. A wet foam elution flush
is performed post-lysis to ensure highly efficient and rapid
removal of lysed material from the lysis system. The subsequent
volume of approximately 1 mL of lysed material is then be processed
in Cartridge B 540.
[0201] Cartridge B 540 operation will essentially be identical to
that of Cartridge A 510 with the exception that it will only have
three membrane stages. In Stage 1 544 the cellular debris created
during the lysis process will be removed. Stage 2 548 will capture
nucleic acids. Stage 3 will capture proteins 550.
[0202] A detailed 24-step process diagram for a single cartridge
fractionation/concentration instrument operation is provided in
FIGS. 8A-8X. The figures clearly demonstrate the action at each
step. They will be summarized here.
[0203] The initial state is shown in FIG. 8X as the conclusive
step, and indicates that: [0204] All valves are closed [0205]
Syringe is homed [0206] Rotary valve is at position 1 (waste)
[0207] The cell is filled with NaOH storage solution [0208] The
sample has been placed in the feed reservoir
[0209] Step 1 is shown in FIG. 8A and indicates that: [0210] The
user is prompted to: "Place a waste container under the retentate
ports" and press "OK" [0211] The rotary valve rotates CW to
position 6 (NaOH reservoir)
[0212] Step 2 is shown in FIG. 8B and indicates that: [0213] The
syringe draws 3 mL of NaOH
[0214] Step 3 is shown in FIG. 8C and indicates that: [0215] The
Rotary valve rotates CCW to position 2 (NaOH inlet) [0216] The
Humic Stage Iso. valves open [0217] The syringe slowly pushes all 3
mL of NaOH through the cell (.about.6 mL/min)
[0218] Step 4 is shown in FIG. 8D and indicates that: [0219] The
syringe completes its stroke [0220] The following valves change
state simultaneously: [0221] Bypass Valves 1-5 open [0222] The
isolation valves open [0223] The Humic Stage Iso. valves close
[0224] Step 5 is shown in FIG. 8E and indicates that: [0225] The
Gas Valve pulses to force the NaOH out the retentate ports
[0226] Step 6 is shown in FIG. 8F and indicates that: [0227] The
foam valve pulses several times to rinse the cell
[0228] Step 7 is shown in FIG. 8G and indicates that: [0229] The
Gas Valve pulses to push out the rest of the foam
[0230] Step 8 is shown in FIG. 8H and indicates that: [0231] The
user is prompted to: "Place a sample container under the retentate
ports" and press "Ok" [0232] The following valves change position
simultaneously: [0233] The Isolation Valves close [0234] The
Feed/Perm Iso. Valves open [0235] The Humic Stage Iso. Valves open
[0236] The syringe draws its full volume
[0237] Step 9 is shown in FIG. 8I and indicates that: [0238]
Diagnostic: The cell should now be at a full vacuum, from now until
Step 12, the pressure should not increase by a significant amount.
The user should be prompted if it is beyond the limit. [0239] The
following valves change position simultaneously: [0240] Filter
Bypasses 1-5 close [0241] Humic Stage Iso. Valves close [0242]
Rotary valve rotates CCW to position 1 (waste) [0243] The syringe
expels its full volume
[0244] Step 10 is shown in FIG. 8J and indicates that: [0245]
Rotary valve rotates CW to position 5 (Feed Reservoir) [0246] The
syringe draws in the feed sample
[0247] Step 11 is shown in FIG. 8K and indicates that: [0248] The
Feed Fluid Sensor sees no fluid [0249] The syringe draws and
additional 10 mL of air
[0250] Step 12 is shown in FIG. 8L and indicates that: [0251] The
rotary valve rotates CCW to position 4 (blocked) [0252] The syringe
draws full volume
[0253] Step 13 is shown in FIG. 8M and indicates that: [0254] The
rotary valve rotates CCW to position 3 (Cell inlet) [0255] The
syringe starts driving the feed sample at the pressure setpoint
[0256] Step 14 is shown in FIG. 8N and indicates that: [0257] The
Inlet Fluid Sensor sees no fluid [0258] The first stage locks up
and the syringe must stop to prevent exceeding the pressure
setpoint
[0259] Step 15 is shown in FIG. 8O and indicates that: [0260] As
each stage locks up, the Bypass valve for that stage is opened
allowing air to pass around the filter
[0261] Step 16 is shown in FIG. 8P and indicates that: [0262] After
the final Filter Bypass valve has been opened, the pressure will
drop rapidly to ambient [0263] The syringe continues its stroke to
expel its full volume
[0264] Step 17 is shown in FIG. 8Q and indicates that: [0265] The
syringe completes its stroke [0266] All of the Bypass valves close
[0267] The Feed/Perm Iso. valves close [0268] The Isolation valves
open
[0269] Step 18 is shown in FIG. 8R and indicates that: [0270] The
Foam Valve pulses to elute the cell
[0271] Step 19 is shown in FIG. 8S and indicates that: [0272] The
Gas Valve pulses to push out the remaining foam [0273] The user is
prompted: "Elute again" or "Complete run" [0274] If "Elute again";
repeat steps 18 and 19 [0275] If "Complete run"; continue to step
20 [0276] Step 1 is shown in FIG. 8A and indicates that:
[0277] Step 20 is shown in FIG. 8T and indicates that: [0278] The
following valves change position simultaneously: [0279] The
Isolation Valves close [0280] The Filter Bypass Valves 1-5 open
[0281] The Feed/Perm Iso. Valves open [0282] The Humic Stage Iso.
Valves open [0283] After a short pause, the syringe draws its full
volume
[0284] Step 21 is shown in FIG. 8U and indicates that: [0285] The
rotary valve rotates CCW to position 1 (waste) [0286] The Feed/Perm
Iso. Valves close [0287] The Filter Bypass Valves 1-5 close [0288]
The syringe expels it's full volume
[0289] Step 22 is shown in FIG. 8V and indicates that: [0290] The
rotary valve rotates CW to position 6 (NaOH reservoir) [0291] The
syringe draws 4 mL
[0292] Step 23 is shown in FIG. 8W and indicates that: [0293] The
rotary valve rotates CCW to position 2 (NaOH inlet) [0294] The
syringe slowly pushes the NaOH into the cell (.about.6 mL/min)
[0295] 3 mL of the fluid fills the inside of the cell, while the
additional 1 mL back flushes the humic stage and goes to waste
[0296] Step 24 is shown in FIG. 8X and indicates that: [0297] The
Humic Stage Iso. Valves close [0298] The rotary valve rotates CCW
to position 1 (waste) [0299] The system resets
[0300] The foam extraction process is summarized below. Sample
extraction can be performed into a small volume using foam made
from the extraction surfactant. This procedure cleans the
concentrator, while simultaneously enhancing extraction efficiency
and allowing for greatly reduced retentate volumes. A small volume
of liquid can be used to create a large volume of foam. Since the
boundaries of the bubbles present in the foam must remain intact to
remain a foam, the boundaries of the bubbles at the interface of
the filter and the extraction foam must always be touching. As the
foam sweeps tangentially across the surface of the filters, it
sweeps the concentrate through the device. When the foam is
extracted from the device and collapses, the remaining product is a
small volume of liquid. This volume can be in a range of less than
5 microliters to 1 milliliter. In its simplest form, the foam may
be made in a separate container, and then injected to sweep the
sample from the concentrator into the sample collection port.
However, the use of a sample loop to measure the amount of liquid
used to make the foam is preferred in order to generate samples of
consistent size. In addition to surfactant foams that are generated
by mixing air and a surfactant solution the foam may also be
generated with a carbonated surfactant solution. Following
carbonation, the solution is agitated by dispensing through an
orifice, frit, filter, or capillary tube. The surfactant foam
extraction methods described here can also be used for extraction
and cleaning of other collection surfaces in aerosol samplers and
collectors. The use of foam to extract these surfaces can provide a
significant increase in extraction efficiency and significant
decrease in final sample volume. Foam made using pressurized carbon
dioxide has been shown in our experiments to be compatible with
collection of viable Bacillus atrophaeus spores. A US Army Natick
Research and Development Engineering Center report,
Natick/TR-94/019, also indicates that Bacillus stereothermophilus
spore suspensions in buffered carbonated solutions were not harmed,
but that germination was inhibited. This inhibition was reversed
upon plating for enumeration. It is also known that carbon dioxide
inhibits the growth of many microorganisms. This fact has been
exploited in preventing bacterial food spoilage in food by using
modified atmosphere packing (MAP, e.g., Baker, R. C., et. al.,
1986, Effect of an elevated level of carbon dioxide containing
atmosphere on the growth of spoilage and pathogenic bacteria at 2,
5, and 13 C. Poult. Sci. 65: 729-737). The inventors believe, based
on data contained in the referenced report, that storage of the
extraction buffer under carbon dioxide pressure will preserve the
extraction fluid from growth of contaminants. Further, since the
foam generation method is driven by the evolution of gas from the
dissolved state in the surfactant extraction fluid, it continues to
generate new bubbles as old bubbles burst during passage though the
fiber. The energy of the bursting bubbles assists in extracting
particles from the fiber filter into the reduced-volume sample. The
majority of the bubbles in the extraction foam burst soon after
release from the extraction cell, resulting in a much smaller
volume sample, which is essentially liquid in nature.
[0301] This application further incorporates by reference herein in
their entirety all of the following applicant-owned applications,
which disclose various techniques of foam elution, as discussed in
the present disclosure Ser. No. 13/368,197; 12/814,993; 12/882,188;
12/883,137; 13/028,897. Such techniques are incorporated by
reference in this application.
[0302] The foregoing disclosure of the exemplary embodiments of the
present subject disclosure has been presented for purposes of
illustration and description. It is not intended to be exhaustive
or to limit the subject disclosure to the precise forms disclosed.
Many variations and modifications of the embodiments described
herein will be apparent to one of ordinary skill in the art in
light of the above disclosure. The scope of the subject disclosure
is to be defined only by the claims appended hereto, and by their
equivalents.
[0303] Further, in describing representative embodiments of the
present subject disclosure, the specification may have presented
the method and/or process of the present subject disclosure as a
particular sequence of steps. However, to the extent that the
method or process does not rely on the particular order of steps
set forth herein, the method or process should not be limited to
the particular sequence of steps described. As one of ordinary
skill in the art would appreciate, other sequences of steps may be
possible. Therefore, the particular order of the steps set forth in
the specification should not be construed as limitations on the
claims. In addition, the claims directed to the method and/or
process of the present subject disclosure should not be limited to
the performance of their steps in the order written, and one
skilled in the art can readily appreciate that the sequences may be
varied and still remain within the spirit and scope of the present
subject disclosure.
* * * * *
References