U.S. patent application number 15/056870 was filed with the patent office on 2019-06-27 for toxin activity assays, devices, methods and systems therefor.
The applicant listed for this patent is Sandia Corporation. Invention is credited to Chung-Yan Koh, Ulrich Y. Schaff, Gregory Jon Sommer.
Application Number | 20190195865 15/056870 |
Document ID | / |
Family ID | 55588927 |
Filed Date | 2019-06-27 |
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United States Patent
Application |
20190195865 |
Kind Code |
A9 |
Koh; Chung-Yan ; et
al. |
June 27, 2019 |
TOXIN ACTIVITY ASSAYS, DEVICES, METHODS AND SYSTEMS THEREFOR
Abstract
Embodiments of the present invention are directed toward
devices, system and method for conducting toxin activity assay
using sedimentation. The toxin activity assay may include
generating complexes which bind to a plurality of beads in a fluid
sample. The complexes may include a target toxin and a labeling
agent, or may be generated due to presence of active target toxin
and/or labeling agent designed to be incorporated into complexes
responsive to the presence of target active toxin. The plurality of
beads including the complexes may be transported through a density
media, wherein the density media has a lower density than a density
of the beads and higher than a density of the fluid sample, and
wherein the transporting occurs, at least in part, by
sedimentation. Signal may be detected from the labeling agents of
the complexes.
Inventors: |
Koh; Chung-Yan; (Dublin,
CA) ; Schaff; Ulrich Y.; (Livermore, CA) ;
Sommer; Gregory Jon; (Livermore, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Sandia Corporation |
Albuquerque |
NM |
US |
|
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20160178619 A1 |
June 23, 2016 |
|
|
Family ID: |
55588927 |
Appl. No.: |
15/056870 |
Filed: |
February 29, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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14157278 |
Jan 16, 2014 |
9304128 |
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15056870 |
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61759486 |
Feb 1, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/54306 20130101;
G01N 2333/31 20130101; G01N 2333/924 20130101; G01N 2333/245
20130101; G01N 2333/70539 20130101; G01N 33/5304 20130101 |
International
Class: |
G01N 33/53 20060101
G01N033/53 |
Goverment Interests
STATEMENT REGARDING RESEARCH & DEVELOPMENT
[0002] This invention was developed under Contract
DE-AC04-94AL85000 between Sandia Corporation and the U.S.
Department of Energy. The U.S. Government has certain rights in the
invention.
Claims
1. An apparatus for conducting a toxin activity assay, the
apparatus comprising: a substrate, wherein the substrate at least
in part defines a channel; a fluid sample contained in the channel,
wherein the fluid sample includes a plurality of beads having
complexes formed thereon by action of an active toxin, individual
ones of the complexes comprising a capture agent and a labeling
agent; a detection region coupled to the channel and defined at
least in part by the substrate and containing a density media,
wherein the density media has a density higher than a density of
the fluid sample and lower than a density of the plurality of
beads; and wherein the channel and detection region are configured
to transport the plurality of beads in the fluid sample from the
channel through the density media responsive to a centrifugal
force, and wherein at least a portion of the free labeling agent is
restricted from transport through the density media.
2. The apparatus of claim 1, wherein said complex further comprises
a target analyte.
3. The apparatus of claim 2, wherein the target analyte comprises a
SEB.
4. The apparatus of claim 1, wherein the active toxin is Ricin.
5. The apparatus of claim 1, wherein the active toxin is Shiga-like
toxin.
6. The apparatus of claim 1, wherein the active toxin is a SEB.
7. The apparatus of claim 1, wherein the beads comprise silica
beads.
8. The apparatus of claim 7, wherein the beads are linked to major
histocompatibility complex II.
9. The apparatus of claim 7, wherein the beads are linked to a DNA
or RNA fragment.
10. The apparatus of claim 9, wherein the DNA or RNA fragment
comprises a Ricin and/or Sarcin loop.
11. The apparatus of claim 1, wherein the labeling agent comprises
Apurinic/apyrimidinic (AP) endonuclease (APE1) enzyme.
12. The apparatus of claim 1, wherein the labeling agent comprises
a stained immortalized T-cell line.
13. The apparatus of claim 12, wherein the immortalized T-cell line
comprises Jurkat cells.
14. The apparatus of claim 13, wherein the Jurkat cells are
fixed.
15. The apparatus of claim 14, wherein the Jurkat cells are stained
by acrinidine orange.
16. The apparatus of claim 1, wherein the density media has a
density less than a density of the plurality of beads but greater
than the fluid sample.
17. A system for conducting a toxin activity assay, the system
comprising: a microfluidic disk comprising: a substrate, wherein
the substrate at least in part defines a channel; a fluid sample
contained in the channel, wherein the fluid sample includes a
plurality of beads having complexes formed thereon by action of an
active toxin, individual ones of the complexes comprising a capture
agent and a labeling agent, wherein the fluid sample further
includes free labeling agent; a detection region coupled to the
channel and defined at least in part by the substrate and
containing a density media, wherein the density media has a density
higher than a density of the fluid sample and lower than a density
of the plurality of beads; and wherein the channel and detection
region are configured to transport the plurality of beads in the
fluid sample from the channel through the density media responsive
to a centrifugal force, and wherein at least a portion of the free
labeling agent is restricted from transport through the density
media; a motor coupled to the microfluidic disk, the motor
configured to receive a motor control signal and spin the
microfluidic disk responsive to the motor control signal; a
detection module positioned to detect a signal from labeling agents
included in the complexes, wherein the detection module is
configured to generate an electronic detection signal based, at
least in part, on the signal from the labeling agent; and a
processing device coupled to the motor and the detection module,
wherein the processing device is configured to generate the motor
control signal and provide the motor control signal to the motor,
and wherein the processing device is further configured to receive
the electronic detection signal from the detection module.
18. The system of claim 17, wherein the signal from the labeling
agents comprises an optical signal and wherein the detection module
comprises a laser and photomultiplier or a laser and photodiode.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional application of, and
discloses subject matter that is related to subject matters
disclosed in, co-pending parent application U.S. Ser. No.
14/157,278, filed Jan. 16, 2014 and entitled "TOXIN ACTIVITY
ASSAYS, DEVICES, METHODS AND SYSTEMS THEREFOR" which claimed
benefit under 35 U.S.C. 119(e) to U.S. provisional patent
application Ser. No. 61/759,486, entitled "METHOD FOR DETECTION OF
TOXIN ACTIVITY IN MICROFLIUDIC DISKS" filed Feb. 1, 2013. The
present application claims the priority of its parent application,
which is incorporated herein by reference in its entirety for any
purpose.
TECHNICAL FIELD
[0003] Embodiments of the invention relate generally to assay
systems and examples include methods, systems, and apparatus for
conducting assays, including the detection and/or quantification of
active toxin such as but not limited to Ricin toxin, Shiga-like
toxins (SLT), and Staphylococcal Enterotoxin B (SEB).
BACKGROUND
[0004] Ricin, Shiga-like toxins (SLT) and Staphylococcal
enterotoxin B (SEB) have either been used as bioterrorism agents or
are considered a bioterrorism threat because of their extreme
toxicity and ease of administration. These toxins can be easily
administered by inhalation, injection or ingestion. In the event of
a mass exposure to biological toxins, identification of the agent
in question is important for accurate diagnostic assessment of
affected patients. It may also, however, be important to determine
the fraction of the toxin which is still active; for example, if a
significant fraction of the toxin is inactive, a treatment may not
be as aggressive as it would be when a large fraction of the toxin
is active. Distinguishing between active and inactive toxin may be
advantageous because of the possibility that genetically engineered
toxins, including the enzymatic portion of the toxin and a binding
domain of another protein, can be used as a bioweapon agent, and
may not be captured by traditional qualitative toxin detection
tests.
[0005] Ricin is a highly toxic protein produced by Ricinus communis
or castor bean plant. It is a category B agent, under the
Biological Select Agents or Toxins, as defined by the United States
Department of Human and Health Services. The major symptoms of
ricin poisoning depend on the route of exposure and the dose
received, though many organs may be affected in severe cases. The
likely symptoms of Ricin inhalation include respiratory distress
(difficulty breathing), fever, cough, nausea, and tightness in the
chest. Finally, low blood pressure and respiratory failure may
occur, leading to death. Swallowing of Ricin would likely lead to
vomiting and diarrhea. Severe dehydration may also result. Other
signs or symptoms may include seizures, and blood in the urine.
Within several days, the person's liver, spleen, and kidneys might
stop working, and the person could die. Ricin is unlikely to be
absorbed through normal skin. Death from ricin poisoning could take
place within 36 to 72 hours of exposure, depending on the route of
exposure (inhalation, ingestion, or injection) and the dose
received.
[0006] Shiga-like toxins (SLTs) are a class of toxins produced by
pathogenic Escherichia coli strains. They cause hemolytic uremic
syndrome in humans, which may lead to death.
[0007] Currently, there are no portable quantitative activity
assays available for determining activity of Ricin and Shiga-like
toxins. The mechanism of action of these toxins generally does not
cause a break in nucleic acid phosphodiester backbone, making it
difficult to determine activity. Accordingly, available assays are
qualitative in nature, only determining presence or absence of
these toxins in a sample, without giving any information regarding
their activity. Further, the few quantitative assays that are
available include tedious processes and steps. For instance,
cell-free translation assays for determining activity of Ricin
toxin require cell-extracts that provide transcriptional and
translational molecular machinery including RNA polymerases for
mRNA transcription, ribosomes for polypeptide translation, tRNA and
amino acids, enzymatic cofactors and energy source, and cellular
components essential for protein folding, while cytotoxicity assays
for determining biological activity require bacterial or tissue
culture cell. Alternatively, mass-spectrometry may be used for
detecting free adenine released in a sample after Ricin attack on
ribosomes, or HPLC-ESI-MS is used for detecting Ricinine, a marker
of Ricin. The processes involving mass-spectrometry suffer from
background noise and reduced sensitivity due to presence of
interfering components in a sample; not to mention cumbersome
equipment that is not portable.
[0008] Not only are these processes labor-intensive, they are also
time-intensive. For instance, the rapid detection tests used by
Center for Disease Control and Prevention's Laboratory Response
Network take 6-8 hours, while the toxin activity tests take about
48 hours. Although there have been reports of new detection assays
that only take 1-2 hours, these assays are only qualitative in
nature and do not give any information on activity of the toxin.
Staphylococcal enterotoxin B (SEB) is an enterotoxin produced by
the bacterium Staphylococcus aureus. It is a common cause of food
poisoning, with severe diarrhea, nausea and intestinal cramping
often starting within a few hours of ingestion. SEB is classified
as an incapacitating agent because in most cases aerosol exposure
does not result in death but in a temporary, though profoundly
incapacitating, illness lasting as long as 2 weeks. SEB is a
superantigen, which causes massive nonspecific activation of immune
system causing release of large amounts of cytokines that lead to
significant inflammation.
[0009] Currently, there are no assays available for determining
activity of SEB. The assays available, such as enzyme-linked
immunosorbent assays (ELISA), chemiluminescence (ECL), and
polymerase chain reaction (PCR), are quantitative in nature and
only aid in detection of SEB.
[0010] Microfluidic systems, including "lab on a chip" or "lab on a
disk" systems continue to be in development. See, Lee, B. S., et.
al., "A fully automated immunoassay from whole blood on a disc,"
Lab Chip 9, 1548-1555 (2009) and Madou, M. et. al., "Lab on a CD,"
Annu. Rev. Biomed. Engr. 8, 601-628 (2006), which articles are
hereby incorporated by reference in their entirety for any
purpose.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 is a flowchart illustrating method for conducting a
Ricin/SLT activity assay in accordance with embodiments of the
present invention.
[0012] FIGS. 2A and 2B are schematic illustrations of a Ricin/SLT
activity assay in accordance with embodiments of the present
invention.
[0013] FIG. 3 is a schematic illustration of a detection region of
a fluid-holding device before and after sedimentation in accordance
with an embodiment of the present invention.
[0014] FIG. 4 is a flowchart illustrating method of conducting a
SEB activity assay in accordance with embodiments of the present
invention.
[0015] FIGS. 5A and 5B are schematic illustrations of a SEB
activity assay in accordance with embodiments of the present
invention.
[0016] FIG. 6 is a schematic illustration of a microfluidic disk
arranged in accordance with embodiments of the present
invention.
[0017] FIGS. 7A, 7B and 7C are schematic illustrations of an assay
area 620 of microfluidic disk in accordance with embodiments of the
present invention.
[0018] FIGS. 8-10 are schematic illustrations of a detection region
containing a sample fluid and a density media in accordance with an
embodiment of the present invention.
[0019] FIG. 11 is a schematic illustration of a system according to
an embodiment of the present invention.
[0020] FIG. 12 shows a dose response curve generated using serial
dilutions of Ricin in accordance with embodiments of the present
invention.
[0021] FIG. 13 shows a dose response curve generated using serial
dilutions of SLT in accordance with embodiments of the present
invention.
[0022] FIG. 14 shows a dose response curve generated using serial
dilutions of SEB in accordance with embodiments of the present
invention.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0023] Certain details are set forth below to provide a sufficient
understanding of embodiments of the invention. However, it will be
clear to one skilled in the art that embodiments of the invention
may be practiced without various of these particular details. In
some instances, well-known chemical structures, chemical
components, molecules, materials, microfluidic components,
electronic components, electronic circuits, control signals, timing
protocols, and software operations have not been shown in detail in
order to avoid unnecessarily obscuring the described embodiments of
the invention.
[0024] Embodiments of the present invention include systems,
apparatuses, and methods for detecting and/or quantifying Ricin,
SLTs and SEB toxin in a sample. As mentioned above, existing
methods for detecting these toxins are generally either limited to
only qualitative detection, and not activity determination, or are
cumbersome and time-intensive. Examples according to the current
invention include analysis of target analytes including toxins such
as but not limited to Ricin, SLTs or SEB. References will be made
herein to and examples given of applications targeting Ricin, SLTs
or SEB, but it should be understood that in other examples, other
toxins may also be targeted for detection and/or quantification. In
examples according to the present invention, the presence of active
toxin may be detected and/or quantified. Active toxin generally
refers to toxin that is able to act on its designated target or
targets. Active toxin generally does not include toxin which may be
present but, for whatever reason, is unable to act (e.g. cleave or
bind) on its designated target or targets.
[0025] Assays described herein may be conducted using systems and
devices that utilize sedimentation to perform assays. For example,
co-pending U.S. application Ser. No. 12/891,977, filed Sep. 28,
2010, entitled "Devices, systems, and methods for conducting
sandwich assays using sedimentation," is hereby incorporated by
reference in its entirety for any purpose. The aforementioned
application includes examples of the formation of complexes
including a capture agent, target analyte, and labeling agent on
sedimentation particles. Thus, target analytes may be separated
from sample by affinity with a capture agent, and the sedimentation
particles passed through a density medium to pellet out at a
detection region of a microfluidic disk. Examples of systems and
devices described in the aforementioned application may be utilized
to conduct assays described herein.
[0026] Examples of the present invention include methods, systems,
and devices, for performing assays to detect ricin, shiga-like
toxins, or both. Ricin generally acts by inhibiting protein
synthesis. It is approximately 64 kDa in size and is composed of
two chains joined by a single disulfide bond. The A chain is
generally responsible for Ricin's catalytic enzymatic activity,
while the B chain is generally responsible for binding to cell
surface receptors on the cell membrane and facilitating entry of
the toxin in to the cytosol. Ricin generally inhibits protein
synthesis by inactivating ribosomes by attacking the sarcin-ricin
loop. This loop is a highly conserved sequence of nucleotides
generally found in the 28S RNA of the large subunit of ribosomes.
It has a conformation of a loop and is generally cleaved by both
.alpha.-sarcin and ricin. Ricin generally acts by specifically and
irreversibly hydrolyzing the N-glycosidic bond of the single
adenine residue at position 4324 (A4324) in the sarcin-ricin loop
within the 28S rRNA, releasing the base, but leaving the
phosphodiester backbone of the RNA intact. Inactive ricin may be
unable to perform this action. The process of attacking and
releasing A4324 is generally referred to as depurination. The
sarcin-ricin loop may be important in binding elongation factors
during protein synthesis; however, depurination by Ricin may
prevent this binding causing inactivation of ribosome, resulting in
toxicity by inhibition of protein synthesis.
[0027] Shiga-like toxins have a similar mechanism of action as
Ricin; they also generally act by depurinating A4324 and thereby
inhibiting protein synthesis. Since both ricin and shiga-like
toxins act by similar mechanisms, generally the same activity
assays may be utilized for both Ricin and SLTs. Further, there may
be other toxins that also act by attacking highly conserved
sequences of different conformations. The same activity assays may
also be utilized for such toxins.
[0028] In an example of an activity assay suitable for detection of
active ricin, SLTs or combinations thereof, depurination caused by
the toxins may be used to determine their activity. Generally, a
labeled enzyme may be used to bind to a site created by an active
toxin attack. For example, a fluorescently-labeled human
Apurinic/apyrimidinic (AP) endonuclease (APE1) enzyme may be used
to bind to the abasic site (i.e., a site without a base, for
example, without A4324) left after an attack by the toxins to
detect depurination, hence the activity of the toxin. The enzyme
may generally be labeled with quantum dots (e.g. QDot 585 from
Molecular Probes), alexa fluor 647 or 10 nm latex particles loaded
with dye. Typically, APEI participates in the DNA base excision
repair (BER) pathway by nicking the phosphodiester backbone at an
AP site (a site without a base, for example, without A4324) through
acyl substitution after a DNA glycosylase removes a damaged or
inappropriate base. This process is mediated by Mg.sup.2+ ions,
which stabilize the AP site and cause release of APE1 from the site
to allow for other enzymes to continue with the BER pathway. In
ricin, SLT, or both, activity assays, binding of fluorescently
labeled APE1 is desired while preventing enzyme turnover. This is
achieved, for example, by depriving APE1 of Mg.sup.2+ ions. Thus,
APE1 binds to the damaged DNA substrate without acting to complete
the repair, leading to detection of depurination caused by
Ricin/SLTs activity. Another advantage of using APE1 in detection
assays is its higher affinity for an abasic site. Unlike other
enzymes that may recognize an abasic site, APE1 is involved in the
BER pathway, which makes it a very strong candidate for detection
of depurination.
[0029] FIG. 1 is a flowchart illustrating a method for conducting
an activity assay for ricin, SLTs, or combinations thereof, in
accordance with embodiments of the present invention. In block 101,
an active toxin attacks a loop linked to a bead causing
depurination of the DNA substrate, giving rise to an abasic site.
In block 102, a labeling agent attaches to the abasic site, forming
a complex. The complexes may be generated on sedimentation
particles (e.g., beads) in a fluid sample. The complexes may
include the loop targeted by the toxin and the labeling agent.
Block 102 may be followed by block 103. In block 103, the
sedimentation particles, e.g., the complexes, may be transported
through a density media having a lower density than the beads but
higher than the fluid sample. Gravitational forces generated
naturally or by centrifugation may be used to transport the beads
through the density media. The sedimentation particles accordingly
may sediment out of the fluid sample, forming a concentrated pellet
at a peripheral portion of a chamber. Block 103 may be followed by
block 104. In block 104, a signal may be detected from the labeling
agent of the complexes. In some examples, by separating the
sedimentation particles from the sample fluid using gravitational
forces, the beads are also concentrated, which may eliminate or
reduce a need for amplification of the labeling agent.
[0030] In block 103, complexes including the target analyte (e.g.,
a toxin), and labeling agent may be formed on beads in a fluid
sample. Any sedimentation particles with appropriate surface
properties, including beads, may be used, including but not limited
to, polystyrene beads, silica beads or poly(methyl methacrylate)
(PMMA) beads. Substantially any bead radii may be used. Examples of
beads may include beads having a radius from 150 nanometers to 10
microns.
[0031] In Ricin/SLTs activity assays described herein, the beads
used to implement the method described with reference to FIG. 1 may
have surface modifications to enable attack by ricin, SLT, or
combinations thereof. For example, synthetic DNA stem loop
substrate, e.g., the sarcin-ricin loop, may be covalently linked to
silica beads. A DNA substrate may be chosen instead of an RNA
substrate due, for example, to ability of the toxins to act on DNA,
as well as due, for example, to a greater stability of DNA compared
to RNA. Similarly, the labeling agent may be any suitable agent for
binding to the target abasic site (AP site) and providing a
detection signal. For example, aforementioned APE1 may have a high
affinity for the abasic site and may attach irreversibly to the
site when deprived of Mg.sup.2+ ions. APE1 may be
fluorescently-labeled for optical detection, however colorimetric
or radioactive tags may also be used.
[0032] FIGS. 2A and 2B are schematic illustrations of a Ricin
and/or SLT activity assay in accordance with embodiments of the
present invention. Although a Ricin/SLT activity assay is shown,
any toxin that causes depurination of a DNA or RNA substrate may be
detected and/or quantified in other examples. FIG. 2A illustrates
an overall mechanism of the activity assay. A sarcinricin loop 201
is shown including adenine residue 202 at position 4324 (A4324).
The residue 202 may be attacked by Ricin and/or SLT causing
depurination in the loop 201 resulting in an abasic site 204. This
process, and thus the activity of Ricin and/or SLT, may be detected
by addition of fluorescently labeled APE1 205, which binds to the
abasic site 204 irreversibly in the absence of Mg.sup.2+.
[0033] In FIG. 2B(1), a sample 206 is shown including Ricin and/or
SLT, e.g., Ricin and/or SLT 203. Samples used in Ricin and/or SLT
activity assays described herein may or may not include Ricin, as
assay may be used to detect the presence of Ricin and/orSLT and/or
to quantify the amount of active Ricin and/or SLT present in a
sample. The sample may include any of variety of fluids, including
biological fluids such as serum.
[0034] FIG. 2B(2) is a schematic illustration of a suspension of
sedimentation particles in accordance with an embodiment of the
present invention. In FIG. 2B(2), a particle suspension 207 may
include sedimentation particles (e.g., bead 208) and labeling agent
(e.g., fluorescently labeled APE 1 205). As has been described
above, the sedimentation particles may have sarcin-ricin loop 201
on their surface. The fluorescently labeled APE 1 205 may be
configured to attach to an abasic site left after an attack from a
toxin. As shown in FIG. 2B, the sample and the particle suspension
may be mixed (e.g. by vortexing, pipetting, and/or sonication).
[0035] FIG. 2B(3) is a schematic illustration of attack of Ricin
and/or SLT 203 on residue 202 at position A4324 in a sarcin-ricin
loop 201 conjugated to the surface of bead 208. This attack results
in formation of abasic site 204.
[0036] FIG. 2B(4) is a schematic illustration of complexes that may
be formed in the mixture. The fluorescently labeled APE 1 205 may
form a complex with the abasic-site 204 (in the sarcin-ricin loop
201) containing bead 208. Any number of complexes may be formed on
the bead, with four shown in FIG. 2B(4). The mixture 209 may be
layered over a density medium 210. The density media 210 is
generally a liquid which may have a density lower than a density of
sedimentation particles (e.g. beads) used in an assay and higher
than a density of the fluid sample. The density media may generally
be implemented using a fluid having a density selected to be in the
appropriate range--lower than a density of the beads used to
conduct an assay and higher than a density of the fluid sample. In
some examples, a fluid sample may be diluted for use with a
particular density media. The density media may include a
Percoll.TM. solution, available from GE lifesciences, which may
contain various additives. Examples of density media may include
95% Percoll.TM.. Generally, viscosity and density may be adjusted
by changing a composition of the media. The mixture 209 may be
introduced into a fluidic feature 211 (e.g. microfluidic device,
including those described herein, vial or other fluid-containing
structure) such that the mixture 209 is next to or on top of the
density media 210. Before or after layering, the mixture may be
incubated to allow for formation of complexes. The density media
may have a density that is greater than the sample but less dense
that the sedimentation particles (e.g. bead 208). Sedimentation
forces (e.g. centrifugal or gravitational) may be applied to the
mixture. For example, a disk containing the fluidic feature
containing the mixture may be spun to apply a centrifugal
force.
[0037] FIG. 2B(5) is a schematic illustration of the fluidic
feature following sedimentation. The sedimentation particles,
including the bead 208 may be transported through the density media
210 responsive to the sedimentation forces and may form a pellet
212 at an end of the structure. As the sedimentation particles
(e.g. beads) are transported through the density media, the flow
may wash the particles, removing unbound material and improving the
detection of the assay. Sample and any unbound label may not be
transported through the density media 210 and may remain within the
sample or at an interface between the density media and the
sample.
[0038] In some examples, it may be of interest to directly measure
active toxin levels in whole blood samples. FIG. 3 is a schematic
illustration of a detection region 330 of a fluid-holding device
(e.g., microfluidic device, disk) before and after sedimentation in
accordance with an embodiment of the present invention. A whole
blood sample 302 including red blood cells 305 and silica beads 315
may be introduced to the detection region 730 next to or over a
density media 310. Although not shown in FIG. 3, the silica beads
315 may be modified with a substrate such as a sarcin-ricin loop or
MHC II conjugated to the beads. This may lead to effective binding
of an active toxin in the fluid sample 302 to form complexes or
formation of complexes responsive to the presence of active toxins,
as generally described herein. The density media 310 may have a
density greater than the red blood cells 305, but less than that of
the beads 315.
[0039] Generally, blood cells may have a density less than or equal
to 1.095 g/cc, and the silica beads may have a density of about
2.05 g/cc. Accordingly, the density media 510 may have a density of
between about 1.095 g/cc and 2.05 g/cc. In one example, the density
media 510 has a density of 1.11 g/cc.
[0040] Sedimentation described herein may occur under the influence
of a natural gravitational force, such as by allowing the assay to
sit, unpowered, under the influence of a gravitational force.
Sedimentation may also occur using centrifugal force, such as by
spinning a microfluidic disk. For examples, silica beads on the
order of 10-30 microns in diameter may sediment in minutes under a
normal gravitational force. Following sedimentation, as shown in
FIG. 3, the blood cells 305 may be prohibited from transport
through the denser density media 310. The beads 315, however, may
be transported through the density media 310 to a detection
location, and active toxin may be tested using signal from a
labeling agent, as described above.
[0041] Ricin and/or SLT activity assays conducted in accordance
with embodiments of the present invention may accordingly separate
interfering matrix components from the active Ricin and/or SLT in
complexes. For example, inactive Ricin and/or SLT or other
components in a sample may not cause depurination of the DNA (or
RNA) substrate covalently linked to surface of silica beads. The
lack of depurination may prevent attachment of fluorescently
labeled APE1 to the substrate, and, thus, formation of
substrate-label complexes. Other components and inactive Ricin
and/or SLT may not cause formation of complexes, and may not travel
through the density media due to the density of the particle being
less than the media. Accordingly, sensitivity of determination of
active Ricin and/or SLT may be improved through use of
sedimentation assays described herein. The sensitivity of detection
and/or quantification may also be improved relative to standard
techniques by determination and/or quantification of the active
Ricin and/or SLT in the sample, concentration in a pellet, and/or
enhancement of fluorescent signal during transport through the
density medium. Still further Ricin and/or SLT assays in accordance
with the present invention may be performed in a relatively short
period of time.
[0042] Examples of the present invention include assays for
detection and/or quantification of active Staphylococcal
enterotoxin B (SEB). SEB is a protein toxin and generally functions
as a superantigen. SEB may be responsible for a number of extensive
pathophysiological changes in humans and mammals and may trigger an
excessive cellular immune response leading to toxic shock. SEB may
be called a superantigen because it interacts with the immune
system to produce an excessive response, activate a very high
percentage of T-cells, which may lead to toxic shock. It is
approximately 24-29 kDa in size. It causes non-specific cross
linking of major histocompatibility complex (MHC) II and T cell
receptors (TCR). This may cause rapid proliferation of T-cells and
production of cytokines, which then lead to significant
inflammation. Exposure to SEB may cause severe diarrhea, vomiting,
cramping, inflammation of skin, eye, fever, headache, and even
toxic shock.
[0043] In an example of SEB activity assay described herein,
crosslinking of MHC II and T-cell receptors by SEB is used to
determine its activity. For example, an immortalized T-cell line,
such as Jurkat cells, which express T-cell receptors on the cell
surface may be used to cross-link with MHC II via SEB. In one
embodiment, fixed Jurkat cells may be used, making them readily
available and precluding maintenance of live cell cultures prior to
the assay. In one embodiment, the Jurkat cells used may be stained
by acrinidine orange. The MHC II may be conjugated to 1 .mu.m
silica microparticles. In presence of SEB a complex may form
between the Jurkat cells and MHC II conjugated to silica
microparticles, through SEB. These complexes may be separated from
unbound Jurkat cells and MHC II-silica microparticles conjugates by
using a density media denser than the Jurkat cells.
[0044] In an example of a SEB activity sedimentation assay, when
Jurkat cells and silica microparticles conjugated to MHC II are
placed together in the presence of SEB, the dense microparticles
may act as a sink, and drive the Jurkat cells down through the
density media by sedimentation force (e.g. gravitational or
centrifugal force). In the absence of active SEB, the Jurkat cells
may stay above the density media.
[0045] FIG. 4 is a flowchart illustrating a method for conducting
SEB activity assay in accordance with embodiments of the present
invention. In block 401, MHC II conjugated to microparticles is
added to a fluid sample. In block 402 a labeling agent, stained
Jurkat cells, expressing TCR, attaches to MHC II via SEB forming a
complex. The complexes may be generated on sedimentation particles
(e.g., beads) in a fluid sample. The complexes may include the
substrate targeted by the toxin and the labeling agent. Block 402
may be followed by block 403. In block 403, the sedimentation
particles, e.g., the complexes, may be transported through a
density media having a lower density than the beads but higher than
the fluid sample. Gravitational forces generated naturally or by
centrifugation may be used to transport the beads through the
density media. The sedimentation particles accordingly may sediment
out of the fluid sample, forming a concentrated pellet. Block 403
may be followed by block 404. In block 404, a signal may be
detected from the labeling agent of the complexes. In some
examples, by separating the sedimentation particles from the sample
fluid using sedimentation forces, the beads are also concentrated,
which may eliminate or reduce a need for amplification of the
labeling agent.
[0046] FIGS. 5A and 5B are schematic illustrations of a SEB
activity assay in accordance with embodiments of the present
invention. Although a SEB activity assay is shown, any toxin that
causes activation of T-cell receptors by cross-linking with MHC II
may be detected and/or quantified in other examples. FIG. 5A
illustrates an example overall mechanism of the activity assay. MHC
II 502 conjugated microparticle 503 (e.g. bead) is shown. Stained
Jurkat cells 501, expressing T-cell receptors (TCR), may be used as
labeling agent. In the presence of SEB 504, a complex may be formed
between stained Jurkat cells 501 and MHC II 502 conjugated
microparticle 503 cross-linked via SEB 504.
[0047] FIGS. 5B(1)-(4) are schematic illustrations of a SEB
activity assay in accordance with embodiments of the present
invention. Although a SEB activity assay is shown, as mentioned
above, any toxin that crosslinks TCR with MHC II may be detected
and/or quantified in other examples. In FIG. 5B(1), a sample 505 is
shown including SEB, e.g. SEB 504. Samples used in SEB activity
assays may or may not include SEB, as the assay may be used to
detect the presence of SEB and/or to quantify the amount of active
SEB present in a sample.
[0048] FIG. 5B(2) is a schematic illustration of a suspension of
sedimentation particles in accordance to an embodiment of the
present invention. In FIG. 5B(2), a particle suspension 506 may
include sedimentation particles (e.g. bead 503) and labeling agents
(e.g. stained Jurkat cells 501 expressing TCR). As has been
described above, the sedimentation particles may be conjugated to
MHC II 502. The stained Jurkat cells 501 expressing TCR may be
stained by acrinidine orange. As shown in FIG. 5, the sample and
the particle suspension may be mixed (e.g. by vortexing, pipeting,
and/or sonication).
[0049] FIG. 5B(3) is a schematic illustration of complexes that may
be formed in the mixture. SEB 504 may form a cross-link between
stained Jurkat cells 501 expressing TCR and MHC II 502 conjugated
to microparticles (e.g. beads 503) resulting in a complex. Any
number of complexes may be formed on the bead, with four shown in
FIG. 5B(3). The mixture 507 may be layered over a density media
508. The density media 508 is generally a liquid which may have a
density lower than a density of sedimentation particles (e.g.
beads) used in an assay and higher than a density of the fluid
sample. The density media may generally be implemented using a
fluid having a density selected to be in the appropriate
range--lower than a density of the beads used to conduct an assay
and higher than a density of the fluid sample. In some examples, a
fluid sample may be diluted for use with a particular density
media. An example of a suitable density media is Percoll.TM.,
available from GE Lifesciences. Particular densities may be
achieved by adjusting a percentage of Percoll.TM. in a solution.
More generally, viscosity and density may be adjusted by changing a
composition of the media. The mixture 507 may be introduced into a
fluidic feature 509 (e.g. microfluidic device, including those
described herein, vial, or other fluid-containing structure) such
that the mixture 507 is next to or on top of the density media 508.
Before or after the layering, the mixture may be incubated to allow
for formation of the complexes. The density media may have a
density that is greater than the sample but less dense than the
sedimentation particles (e.g. bead 503). Sedimentation forces (e.g.
centrifugal or gravitational) may be applied to the mixture. For
example, the feature containing the mixture may be spun to apply a
centrifugal force.
[0050] FIG. 5B(4) is a schematic illustration of the fluid
containing structure following sedimentation. The sedimentation
particles, including the bead 503 may be transported through the
density media 508 responsive to the sedimentation forces and may
form a pellet 510 at an end of the structure. As the sedimentation
particles (e.g. beads) are transported through the density media,
the flow may wash the particles, removing unbound material and
improving the detection of the assay. Sample and any unbound label
may not be transported through the density media 508 and may remain
within the sample or at an interface between the density media and
the sample.
[0051] SEB activity assays conducted in accordance with embodiments
of the present invention may accordingly separate interfering
matrix components from the active SEB the in complexes. For
example, inactive SEB or other components in a sample may not cause
cross-linking of cells expressing TCR and MHC II conjugated to
silica beads. The lack of cross-linking will prevent attachment of
stained cells to the substrate, and, thus, formation of
substrate-label complexes. Other components and inactive SEB may
not cause formation of complexes, and may not travel through the
density media due to the density of the interfering components
being less than the media. Accordingly, sensitivity of
determination of active SEB may be improved. The sensitivity of
detection and/or quantification may also be improved relative to
standard techniques by determination of only the active SEB in the
sample, concentration in a pellet, and/or enhancement of
fluorescent signal during transport through the density medium.
Still further SEB assays in accordance with the present invention
may be performed in a relatively short period of time.
[0052] In some embodiments, methods described herein may be
implemented using a microfluidic disk. FIG. 6 is a schematic
illustration of a microfluidic disk 600 arranged in accordance with
embodiments of the present invention. The microfluidic disk 600 may
include a substrate 610 which may at least partially define regions
of assay areas 620, 621, 622, and 623. The microfluidic disk 600
may include a fluid inlet port 625 in fluid communication with the
assay areas 620, 621, 622, and 623. Each assay area my include any
of a variety of fluidic features and components, including but not
limited to, channels, chambers, valves, pumps, etc. As show in FIG.
6, each assay area includes a mixing chamber and a detection region
connected by a channel, which may contain a valve. During
operation, fluids including sample liquids, density media, and/or
beads suspended in a fluid, may be transported using centrifugal
force from an interior of the microfluidic disk 600 towards a
periphery of the microfluidic disk 600 in a direction indicated by
an arrow 630. The centrifugal force may be generated by rotating
the microfluidic disk 600 in the direction indicated by the arrow
635, or in the opposite direction.
[0053] The substrate 610 may be implemented using any of a variety
of suitable substrate materials. In some embodiments, the substrate
may be a solid transparent material. Transparent plastics, quartz,
glass, fused-silica, PDMS, PMMA and other transparent substrates
may be desired in some embodiments to allow optical observation of
sample within the channels and chambers of the disk 600. In some
embodiments, however, opaque plastic, metal or semiconductor
substrates may be used. In some embodiments, multiple materials may
be used to implement the substrate 610. The substrate 610 may
include surface treatments or other coatings, which may in some
embodiments enhance compatibility with fluids placed on the
substrate 610. In some embodiments surface treatments or other
coatings may be provided to control fluid interaction with the
substrate 610. While shown as a round disk in FIG. 46 the substrate
610 may take substantially any shape, including square.
[0054] In some embodiments, as will be described further below, the
substrate 610 may itself be coupled to a motor for rotation. In
some embodiments, the substrate may be mounted on another substrate
or base for rotation. For example, a microfluidic chip fabricated
at least partially in a substrate may be mounted on another
substrate for spinning. In some examples, the microfluidic chip may
be disposable while the substrate or base it is mounted on may be
reusable. In some examples, the entire disk may be disposable. In
some examples, a disposable cartridge including one or more
microfluidic channels may be inserted into disk or other mechanical
rotor that forms part of a detection system.
[0055] The substrate 610 may generally at least partially define a
variety of fluidic features. The fluidic features may be
microfluidic features. Generally, microfluidic, as used herein,
refers to a system, device, or feature having a dimension of around
1 mm or less and suitable for at least partially containing a
fluid. In some embodiments, 500 .mu.m or less. In some embodiments,
the microfluidic features may have a dimension of around 100 .mu.m
or less. Other dimensions may be used. The substrate 410 may define
one or more fluidic features, including any number of channels,
chambers, inlet/outlet ports, or other features.
[0056] Microscale fabrication techniques, generally known in the
art, may be utilized to fabricate the microfluidic disk 600. The
microscale fabrication techniques employed to fabricate the disk
600 may include, for example, embossing, etching, injection
molding, surface treatments, photolithography, bonding and other
techniques.
[0057] A fluid inlet port 625 may be provided to receive a fluid
(e.g. sample) that may be analyzed using the microfluidic disk 600.
The fluid inlet port 625 may have generally any configuration, and
a fluid sample may enter the fluid inlet port 625 utilizing
substantially any fluid transport mechanism, including pipetting,
pumping, or capillary action. The fluid inlet port 425 may take
substantially any shape. Generally, the fluid inlet port 625 is in
fluid communication with at least one assay area, and may be in
fluid communication with multiple assay areas 620-623 in FIG. 4.
Generally, by fluid communication it is meant that a fluid may flow
from one area to the other, either freely or using one or more
transport forces and/or valves, and with or without flowing through
intervening structures.
[0058] The assay area 620 generally may include one or more
channels in fluid communication with the fluid inlet port 625.
Although four assay areas 620-623 are shown in FIG. 6, generally
any number may be present on the microfluidic disk 600.
[0059] As the microfluidic disk 600 is rotated in the direction
indicated by the arrow 635 (or in the opposite direction), a
centrifugal force may be generated. The centrifugal force may
generally transport fluid from the inlet port 625 into one or more
of the assay areas 620-623.
[0060] Accordingly, the microfluidic disk 600 may be used to
perform assays described herein. Centrifugal forces may be used to
generate sedimentation forces described herein. In other examples,
however, gravity may be used to generate sedimentation forces, and
assays described herein may be conducted in a vial or other
container.
[0061] FIGS. 7A-C are schematic illustrations of an assay area 620
of a microfluidic disk in accordance with an embodiment of the
present invention. The assay area 620 includes a channel 710 in
fluid communication with the fluid inlet port 625. The channel 710
may be in fluid communication with a detection region 730. Another
reservoir 735 may be in fluid communication with the detection
region 730 via a channel 740, which may include or serve as a
valve. The detection region 730 may be implemented as a fluidic
chamber, channel, or reservoir, and detection may occur at an end
of the region. In other examples, detection may not occur in the
detection region 730, but the sample may be transported elsewhere
for detection.
[0062] The detection region 730 and reservoir 735 may generally be
implemented using any size and shape, and may contain one or more
reagents including solids and/or fluids which may interact with
fluid entering and/or exiting the features.
[0063] The detection region 730 may be configured to contain a
density media. Constituents of an appropriate density media are
explained previously. In some embodiments, the density media may
include a detergent, such as Tween 20. The detergent may enhance a
wash function of transport through the density media.
[0064] Sample, sedimentation particles, and labeling agent may
first be mixed and/or incubated in the reservoir 735, as shown in
FIG. 7A, then introduced at a selected time to the detection region
730 by operation of the valve 740. That is, a mixture of sample,
sedimentation particles, and labeling agent suspension may be
present in the reservoir 735 (for example by introducing the
different components separately, or by loading a mixture containing
all components into the reservoir 735). The valve 740 may be closed
to contain the components in the reservoir 735 and allow the
components to incubate to form complexes.
[0065] On opening the valve 740, as shown in FIG. 7B, the mixture
including the sample, beads, and labeling agent, which may have
formed complexes, may be introduced to the detection region 730. In
some examples, the reservoir 735 may not be provided, and the
mixture may be loaded directly into the detection region 730, which
may be a channel or chamber. Detection may take place at an end of
the detection region 730. The detection region 730 may further have
a tapered or other shape at the end of the detection region
730.
[0066] The detection region 730 may be a channel or chamber and may
vary in configuration in accordance with the detection technique
employed, as will be described further below. The detection region
730 may generally be configured to allow for detection of a signal
emitted by labeling agents in a complex. The complex may include
active toxin and labeling agent in embodiments pertaining to toxin
activity assays. In some examples, the complex may not itself
include active toxin, but the complex may have formed due in part
to the presence of active toxin in a sample. The complexes include
sedimentation particles (e.g. microparticles, beads).
[0067] Centrifugal forces may generally be used to transport the
mixture from the inlet port 725 toward the detection region 730.
Additionally, centrifugal forces may be used to transport density
media from the reservoir 735 to the detection region 730. In other
examples, pressure-driven or other flow drivers may be used to load
fluids into the device and transport the mixture to the detection
region 730.
[0068] Incubation of sedimentation particles and labeling agents
with the sample may take place within a microfluidic disk.
Referring again to FIG. 7A, a fluid sample containing a target
analyte, e.g. toxin, may be introduced to the inlet port 625 and
provided to the reservoir 735 through the channel 710. Any of a
variety of suitable fluid samples may be used including, but not
limited to, whole blood, buffer solutions, serum, or other
biological fluid samples. Generally, the fluid sample will include
target analytes to be detected in accordance with embodiments of
the present invention. The fluid sample may contain beads designed
to form complexes responsive to the presence of active toxins
and/or labeling agents designed to be incorporated into complexes
responsive to the presence of active toxins. In other examples, the
beads and/or label agents may be introduced to the fluid sample
within the microfluidic disk 600. For example, a fluid containing
the beads and/or label agents may be provided to a different inlet
port in fluid communication with the channel 710 of FIG. 7. Either
by mixing components or by providing a fluid containing the
components, a sample fluid including beads, target analytes, and
labeling agents, may be transported to the detection region 730 of
FIG. 7. The transport of the sample fluid may occur through any
type of transport mechanism, including centrifugal force,
pressure-driven flow, pumping, or other mechanisms. In other
examples, beads having capture agents on their surface may be
incubated with target analyte and/or labeling agents prior to
introduction to a microfluidic disk. In such an example, complexes
may be formed on beads in a sample fluid prior to providing the
sample fluid to the microfluidic disk.
[0069] The detection region 730 of FIG. 7 may contain density
media, or density media may be transported into the detection
region 730 from another location, such as from the reservoir 735.
The channel 740 may have a width selected to serve as a valve, such
that a spin rate over a threshold amount is required to initiate a
flow of the density media from the reservoir 735 through the
channel 740. In some examples, the channel 740 has a width selected
such that any spin rates used to transport sample into the
reservoir 735 is insufficient to transport the sample into the
detection region 730. A spin rate of the microfluidic disk 600 may
then be increased to initiate or enhance a flow of the sample from
the reservoir 735 into the detection region 730. In this manner,
the channel 740 may function as a valve. Other valve structures
such as wax plugs that melt at a known temperature may be used in
other examples.
[0070] Once the mixture of sample, bead, and labeling agent is in
the detection region 730 above the density media (e.g. closer to
the center of the disk), sedimentation forces may be used to
transport the beads through the density media to form a bead
pellet, as shown in FIG. 7C. If labeling agent is present on the
bead, they may be detected in the concentrated pellet.
[0071] Accordingly, a sample fluid including: 1) beads; 2) target
analytes; and 3) labeling agents may be transported to an interface
with a density media and sedimentation forces used to transport the
beads, along with any bound complexes, through the density media to
form a pellet at the end of the detection region.
[0072] FIG. 8-9 are schematic illustrations of the detection region
730 containing a sample fluid 801 and a density media 802 in
accordance with an embodiment of the present invention. Components
of the sample fluid 801 are shown for the purposes of illustration
beneath the detection region 730 in FIG. 8. In FIGS. 8-9, schematic
views of the sample components, and complex formation, are shown
below the detection region view for ease of illustration. The
sample fluid includes a plurality of beads, including bead 803 with
surface modification (e.g., sarcin-ricin loop or MHC II). The
sample fluid 801 further includes target analytes, such as active
toxin 804, and labeling agent 805.
[0073] The sample fluid may then be incubated. FIG. 9 is a
schematic illustration of the detection region 730 containing the
sample fluid 801 and the density media 802 following an incubation
period. Complexes may form on the bead 803. The target analyte 804
may bind to the bead 803 and labeling agent 805; alternatively, the
target analyte may not bind to the bead 803 and labeling agent 805,
but may cause formation of complexes responsive to the presence of
analyte (active toxins) and/or label agents designed to be
incorporated into complexes responsive to the presence of the
target analyte (not shown in FIGS. 8 and 9). Some unbound, free
labeling agents, however, remain in the sample fluid 801. At times
a little to no centrifugal force may be provided to aid incubation.
Additionally, in some examples, a region of the microfluidic disk
containing the sample fluid may be headed to enhance incubation. In
this manner, complexes may be formed on the beads. As understood in
the art, the amount of labeling agent bound to complexes on the
beads will be generally proportional to the amount of target
analyte in the fluid sample. Any number of complexes may be formed
on the beads, with three complexes shown on the bead 803 in FIG.
9.
[0074] The beads may then be transported through the density media.
The beads are transported through the density media using
centrifugal force, such as that which may be applied by motor,
described further below. Following a period of centrifugal force,
the beads may be concentrated in a detection location. FIG. 10 is a
schematic illustration of the detection region 730 following
transport of beads through the density media. The fluid sample 801
may remain separated from the density media 802, as the density
media 802 may have density higher than that of the sample fluid
801. Free, unbound labeling agent, may remain in the sample fluid
801. Beads 803, including complexes, may be transported through the
density media 802 to a detection location 806. Beads at the
detection location 806 are shown for purposes of illustration under
the detection region 730 in FIG. 10. The bead 803 includes
complexes containing target analyte 804 and labeling agent 805;
alternatively, the bead 803 may include complexes formed in
response to the presence of active toxins and/or label agents
designed to be incorporated into complexes responsive to the
presence of the target analyte (not shown in FIGS. 8 and 9).
However, unbound labeling agent may not be found in the detection
location 806. As shown in FIGS. 8-10, centrifugal force may
accordingly be used to separate complexed beds from a sample fluid
and to concentrate the complexed beads. In this manner, the need
for additional assay may be reduced or eliminated. Signal from
labeling agent of the concentrated beads may be detected from the
detection location 806 using, for example, the detection module
examples described below.
[0075] FIG. 11 is a schematic illustration of a system according to
an embodiment of the present invention. The system 1100 may include
the disk 600 of FIG. 6 with one or more assay areas 620. A motor
1101 may be coupled to the disk 600 and configured to spin the disk
600, generating centrifugal forces. A detection module 1102 may be
positioned to detect signal from labeling agents in a detection
region of the assay area 620. An actuator 915 may be coupled to the
detection module 1102 and configured to move the detection module
along the detection region in some examples. A processing device
1104 may be coupled to the motor 1101, the detection module 102,
and/or the actuator 1103, and may provide control signals to those
components. The processing device 1104 may further receive
electronic signals from the detection module 1102 corresponding to
the labeling agent signals received from the detection module 1102.
All or selected components shown in FIG. 11 may be housed in a
common housing in some examples. Microfluidic disks, which may be
disposable, may be placed on the motor 1101 and removed, such that
multiple disks may be analyzed by the system 1100.
[0076] The motor 1101 may be implemented using a centrifugation
and/or stepper motor. The motor 1101 may be positioned relative to
the detection module 1102 such that, when the disk 600 is situated
on the motor 1101, the disk is positioned such that a detection
region of the assay area 620 is exposed to the detection module
1102.
[0077] The detection module 1102 may include a detector suitable
for detecting signal from labeling agents in complexes that may
include labeling agent. In toxin activity assays (e.g., Ricin, SLT,
and/or SEB) assays, the complexes may include active toxin and
labeling agent or may form in response to the presence of an active
toxin and/or label agent designed to be incorporated into complexes
responsive to the presence of active toxin. The complexes may be
formed on the surface of one or more sedimentation particles (e.g.,
beads), as described further below. The detector may include, for
example, a laser and optics suitable for optical detection of
fluorescence from fluorescent labeling agents. The detection module
may include one or more photomultiplier tubes. In other examples,
other detectors, such as photodiodes or CCD cameras, may be used.
The actuator 1101 may move the detector in some examples where
signal may be detected from a variety of locations of the
microfluidic disk 600.
[0078] The processing device 1104 may include one or more
processing units, such as one or more processors. In some examples,
the processing device 1104 may include a controller, logic
circuitry, and/or software for performing functionalities described
herein. The processing device 1104 may be coupled to one or more
memories, input devices, and/or output devices including, but not
limited to, disk drives, keyboards, mice, and displays. The
processing device may provide control signals to the motor 1101 to
rotate the disk 600 at selected speeds for selected times. The
processing device may provide control signals to the detection
module 1102, including one or more detectors and/or actuators, to
detect signals from the labeling agents and/or move the detector to
particular locations. The processing device may develop these
control signals in accordance with input from an operator and/or in
accordance with software including instructions encoded in one or
more memories, where the instructions, when executed by one or more
processing units, may cause the processing device to output a
predetermined sequence of control signals. The processing device
1104 may receive electronic signals from the detection module 910
indicative of the detected signal from labeling agents. The
processing device 1104 may detect a target analyte and/or calculate
a quantity of a target analyte in a fluid sample based on the
signals received from the detection module 1102. Accordingly, the
processing device 1104 may perform calculations in accordance with
software including one or more executable instructions stored on a
memory causing the processing device to perform the calculations.
Results may be stored in memory, communicated over a network,
and/or displayed. It is to be understood that the configuration of
the processing device 1104 and related components is quite
flexible, and any of a variety of computing systems may be used
including server systems, desktops, laptops, controllers, and the
like.
[0079] Having described examples of micro fluidic disks and systems
in accordance with embodiments of the present invention, methods
for conducting assays will now be described. Some discussion will
also be provided regarding mechanisms for sedimentation and
centrifugation. The discussion regarding mechanisms is provided as
an aid to understanding examples of the present invention, but is
in no way intended to limit embodiments of the present invention.
That is, embodiments of the present invention may not employ the
described mechanism.
[0080] Sedimentation of spheres may occur within a viscous fluid
under the influence of a gravitational field (which may be natural
or induced by centrifugation). The settling velocity of
approximately spherical particles may be described by Stoke's flow
equations:
V s = 2 9 - ( .rho. p - .rho. f ) .mu. gR 2 ; ##EQU00001##
[0081] where Vs is the sedimentation velocity, .mu. is the fluid
viscosity, .rho..rho. is the density of the particle, .rho.f is the
density of the fluid, g is acceleration due to effective gravity,
and R is the particle radius. Note that sedimentation rate scales
with the square of particle radius and therefore a small difference
in radius may form a basis for separation of particles in some
examples, as they may sediment at a different rate. There is also a
linear dependence of sedimentation rate with the difference in
density between the particle and the surrounding fluid, which may
also be an effective mechanism for separation. Accordingly, beads
or other particles may be separated according to their density
and/or radius based on different sedimentation velocities.
Separation of particles using these principles may be referred to
as "rate zonal centrifugation."
[0082] For nanometer scale particles, such as active toxins,
gravitational forces may act in conjunction with Brownian
diffusion, but neither will generally cause motion of these
nanometer scale particles over significant distances during typical
centrifugal conditions (<100,000 g). Accordingly, beads with
affinity for active toxins, due to surface modifications, may be
used to separate active toxins from a fluid sample containing
mixture of other small molecules. By capturing active toxin on the
bead surface, and separating the beads from the remaining sample
using sedimentation (e.g. centrifugal or gravitational) forces, the
need for wash steps may be reduced or eliminated, because unbound
labeling agents and/or other molecules may be dissociated from the
beads by fluid flow.
Example 1
[0083] For ricin and/or shiga-like toxin activity assay, a sample
may be incubated with microparticle-conjugated stem-loop substrates
for 20 or 240 min in 10 mM potassium citrate at pH 4.0. To this
suspension, fluorescently labeled APE1 may be added to a final
concentration of 100 nM, as well as PBS (pH 7.4) with between 50 mM
EDTA and 250 mM EDTA. The fluorescent label may be an organic dye,
such as Alexa Fluor 647 from Molecular Probes, a quantum dot, such
as QDot 585 from Molecular Probes, dye-loaded latex particles, such
as 10 nm FluoSpheres from Life Technologies. The suspension may be
allowed to react for 20 min. After incubation, 4 .mu.L of the
suspension may be added to a channel pre-loaded with density
medium. Density medium may be 95% Percoll and 5% (v/v) concentrate
of additives in distilled water containing 50 mM sodium phosphate,
125 mM EDTA, 0.01% Tween 20 (w/v) and 100 mM sodium chloride. The
channel may be spun at 8000 rpm for 45 s, transporting the
microparticles through the density medium to form a pellet in a
detection area. The pellet may be imaged using a fluorescent
microscope.
[0084] FIG. 12 shows a dose response curve generated using serial
dilutions of Ricin. Concentration of ricin in picomoles (pM) is
plotted on the X-axis against the corresponding fluorescence
intensity measured in relative fluorescence units (RFU) on the
Y-axis. In assay where a sample containing Ricin was incubated with
a labeling agent and modified beads for 20 minutes, the lowest
concentration of ricin that could be detected was 780 pM. However,
in an assay where the incubation period was increased to 240
minutes, the lowest concentration of ricin that could be detected
was 13 pM. As may be seen in FIG. 12, the fluorescence intensity
detected increased with increase in concentration of ricin.
[0085] FIG. 13 shows a dose response curve generated using serial
dilutions of SLT. Concentration of SLT in nanomoles (nM) is plotted
on the X-axis against the corresponding fluorescence intensity
measured in relative fluorescence units (RFU) on the Y-axis. As may
be seen in FIG. 13, the fluorescence intensity detected increased
with increase in concentration of SLT.
Example 2
[0086] For SEB activity assay, MHC II may be immobilized to 1 .mu.m
carboxylic acid functionalized microparticles. Jurkat cells may be
fixed in ice-cold methanol for 5 min, washed in PBS, and stained
with 100 .mu.M acrinidine orange for 10 min. Following which the
cells may be washed in PBS and resuspended to a concentration of
3.times.10.sup.7 cells/mL. To 7 .mu.L of a 5% solid suspension of
MHC II-conjugated microparticles, 7 .mu.L of SEB toxin and 1 .mu.L
of stained Jurkat cells may be added.
[0087] Hela cells may be used as a negative control. HeLa cells may
be treated with trypsin and EDTA for 5 min at 37.degree. C., washed
with PBS, fixed in ice-cold methanol for 5 min, stained with 100
100 .mu.M acrinidine orange for 10 min, and resuspended at a
concentration of 3.8.times.10.sup.7 cell/mL. To 7 .mu.L of a 5%
solids suspension of MHC II-conjugated microparticles 7 .mu.L of
SEB toxin and 1 .mu.L of HeLa cells may be added.
[0088] The channel may be spun at 8000 rpm for 45 s, transporting
the microparticles through the density medium to form a pellet in a
detection area. Resultant bead pellets (for both Jurkat cells and,
negative control, HeLA cells) may be imaged on a fluorescent
microscope using 488 nm excitation and 525 nm emission.
[0089] FIG. 14 shows a dose response curve generated using serial
dilutions of SEB. Concentration of SEB in nanomoles (nM) is plotted
on the X-axis against the corresponding fluorescence intensity
measured in relative fluorescence units (RFU) on the Y-axis. As may
be seen in FIG. 14, the fluorescence intensity detected increased
with increase in concentration of SEB.
* * * * *