U.S. patent application number 15/616740 was filed with the patent office on 2017-09-21 for system and method for detecting components of a mixture including a valving scheme for competition assays.
The applicant listed for this patent is National Technology & Engineering Solutions of Sandia, LLC. Invention is credited to Chung-Yan Koh, Matthew E. Piccini, Anup K. Singh.
Application Number | 20170269072 15/616740 |
Document ID | / |
Family ID | 59257536 |
Filed Date | 2017-09-21 |
United States Patent
Application |
20170269072 |
Kind Code |
A1 |
Koh; Chung-Yan ; et
al. |
September 21, 2017 |
SYSTEM AND METHOD FOR DETECTING COMPONENTS OF A MIXTURE INCLUDING A
VALVING SCHEME FOR COMPETITION ASSAYS
Abstract
Examples are described including measurement systems for
conducting competition assays. A first chamber of an assay device
may be loaded with a sample containing a target antigen. The target
antigen in the sample may be allowed to bind to antibody-coated
beads in the first chamber. A control layer separating the first
chamber from a second chamber may then be opened to allow a
labeling agent loaded in a first portion of the second chamber to
bind to any unoccupied sites on the antibodies. A centrifugal force
may then be applied to transport the beads through a density media
to a detection region for measurement by a detection unit.
Inventors: |
Koh; Chung-Yan; (Dublin,
CA) ; Piccini; Matthew E.; (Belmont, CA) ;
Singh; Anup K.; (Danville, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
National Technology & Engineering Solutions of Sandia,
LLC |
Albuquerque |
NM |
US |
|
|
Family ID: |
59257536 |
Appl. No.: |
15/616740 |
Filed: |
June 7, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
14546876 |
Nov 18, 2014 |
9702871 |
|
|
15616740 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 2200/0684 20130101;
B01L 2300/0803 20130101; B01L 2300/087 20130101; B01L 2300/0874
20130101; G01N 33/54306 20130101; G01N 33/54366 20130101; B01L
2400/0677 20130101; B01L 2400/0683 20130101; B01L 2400/043
20130101; B01L 2200/0647 20130101; B01L 3/502738 20130101; B01L
2400/0409 20130101 |
International
Class: |
G01N 33/543 20060101
G01N033/543; B01L 3/00 20060101 B01L003/00 |
Goverment Interests
STATEMENT REGARDING RESEARCH & DEVELOPMENT
[0002] This invention was made with Government support under
Contract No. DE-NA0003525 awarded by the United States Department
of Energy/National Nuclear Security Administration. The U.S.
Government has certain rights in the invention.
Claims
1. A method of conducting a competition assay for detection of a
target analyte, the method comprising: loading a sample into a
first chamber of an assay device, wherein the first chamber is
loaded with beads coated with antibodies; incubating the sample and
beads to allow formation of complexes of beads and the target
analyte when present in the sample; opening at least a portion of a
control layer dividing the first chamber from a second chamber to
provide a fluidic connection between the first chamber and a first
portion of the second chamber, wherein the second chamber contains
a labeling agent in the first portion of the second chamber and a
density media in a second portion of the second chamber, wherein
the labeling agent is configured to compete for sites on the
antibodies with the target analyte; allowing the labeling agent to
bind to unoccupied sites on the antibodies in the first portion of
the second chamber; transporting the beads through the density
media, wherein the density media has a density lower than a density
of the beads; and detecting signal from labeling agents bound to
the antibodies.
2. The method of claim 1 further comprising transporting the beads
from the first chamber to the second chamber by a pressure gradient
after providing the fluidic connection.
3. The method of claim 1 further comprising mixing the plurality of
beads and the labeling agent after opening at least a portion of
the control layer.
4. The method of claim 3, wherein the mixing is responsive to
moving the assay device.
5. The method of claim 3, wherein said opening at least a portion
of the control layer creates a region including the first portion
of the second chamber having a turbulent flow regime, and wherein
the mixing involves turbulent flow in the first portion of the
second chamber.
6. The method of claim 5, wherein the second portion of the second
chamber is sized to have a laminar flow regime, and wherein moving
the assay device mixes contents of the first portion of the second
chamber without mixing the contents of the second portion of the
second chamber.
7. The method of claim 1, wherein the control layer is formed from
wax, a polymer, a photoresponsive material, a chemoresponsive
material, glass, thermoplastics or combinations thereof.
8. The method of claim 1, wherein opening at least a portion of the
control layer comprises moving one or more pins through the control
layer.
9. The method of claim 8, wherein the first chamber and the second
chamber comprise a first assay set, and wherein the assay device
further comprises multiple assay sets.
10. The method of claim 9, further comprising moving multiple pins,
each corresponding to a respective assay region, through the
control layer simultaneously.
11. The method of claim 10, wherein moving the pins comprises
applying a magnetic force to the pins.
12. The method of claim 1, wherein the target analyte comprises an
antigen and the labeling agent comprises a labeled antigen.
13. The method of claim 1, wherein a strength of the signal
detected is inversely proportional to an amount of the target
analyte in the sample.
14. The method of claim 1 further comprising venting the second
chamber after opening at least a portion of the control layer
through a vent coupled to the second chamber.
15. The method of claim 1, wherein transporting the plurality of
complexes comprises applying a centrifugal force applied by a motor
coupled to the assay device.
16. The method of claim 1, wherein the labeling agent is a
fluorescent labeling agent and the detecting signal comprises
detecting a fluorescent signal.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional of prior application Ser.
No. 14/546,876, filed Nov. 18, 2014, which is hereby incorporated
by reference in its entirety.
TECHNICAL FIELD
[0003] Embodiments of the invention relate generally to measurement
systems and examples include methods, systems and apparatus
including mechanisms for conducting competition assays for the
detection and/or quantification of a target analyte in a sample.
Examples of microfluidic disks including mechanisms for conducting
competition assays are described.
BACKGROUND
[0004] Measurement of samples is important in a number of
industries, such as the chemical and biotechnology industries,
where the concentration of certain components of the sample is of
interest. For example, quantification of biomolecules such as
proteins and nucleic acids from patient samples is an important
area of research and commercial development. Quantification of
biomolecules and other types of samples is typically performed by
optical measurements including fluorescence, luminescence, or
relative light absorption. Portable solutions for these
applications are a large and growing segment of the overall market.
Existing portable solutions typically require specialized
personnel, and may be bulky and time-consuming to operate.
[0005] Measurement systems sometimes perform sandwich ELISA assays
in which two antibodies participate in the assay. This may be
achieved by using a capture antibody to immobilize the antigen on a
solid support and a labeled detection antibody for quantification.
Sandwich ELISA assays generally may be performed in a single
chamber because of the specificity of the monoclonal antibodies,
which typically do not cross-react and do not bind to each other in
the absence of the antigen. While sandwich ELISA assay may be
sufficient for detection of some types of target analytes,
competition ELISA may provide a greater sensitivity for detection
of certain target analytes.
[0006] Competition ELISA generally involves antibodies immobilized
on a solid support. Antigen in the sample may bind to sites on the
antibodies. Following a period of time where antigen in a sample is
allowed to bind, labeled antigen that is selected to bind to the
same or similar sites on the antibodies is introduced. The labeled
antigen "competes" for sites with the antigen in the sample. To the
extent antigen was not present in the sample, sites will remain for
the labeled antigen to bind. In this manner, the amount of signal
in the competition assay may be inversely proportional to the
quantity of antigen in the sample.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1 is a top down schematic illustration of an assay
device arranged in accordance with embodiments of the present
invention.
[0008] FIG. 2 is a top down exploded schematic illustration of an
assay device including multiple layers, according to examples of
the present invention.
[0009] FIG. 3 is a bottom up schematic illustration of an assay
device arranged in accordance with examples of the present
invention.
[0010] FIG. 4 is a bottom up exploded schematic illustration of an
assay device arranged in accordance with examples of the present
invention.
[0011] FIG. 5 is a cross-sectional schematic illustration of an
assay device according to examples of the present invention.
[0012] FIG. 6 is a cross-sectional schematic illustration of an
assay device prior to opening a control layer in accordance with
examples of the present invention.
[0013] FIG. 7 is a cross-sectional schematic illustration of an
assay device after opening a control layer in accordance with
examples of the present invention.
[0014] FIG. 8 is a flowchart depicting a method for performing an
assay, according to examples of the present invention.
[0015] FIG. 9 is a top down exploded schematic illustration of the
first chamber layer, the control layer, and the second chamber
layer in accordance with examples of the present invention.
[0016] FIG. 10 is a schematic illustration of a system for
conducting an assay in accordance with examples of the present
invention.
DETAILED DESCRIPTION
[0017] 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, electronic components, 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.
[0018] Disclosed herein are example embodiments of systems,
apparatuses and methods for detecting and/or quantifying one or
more components of a sample. Examples are described include
mechanisms for conducting competition assays for detection and/or
quantification of a target analyte in the sample. As mentioned
above, existing systems and methods for performing an assay may be
cumbersome and offer limited sensitivity. Therefore, there may be a
need for user friendly systems and methods to perform a competitive
assay quickly, accurately, and at a relatively low cost. Disclosed
systems and methods may be used to perform chemical assays,
biochemical assays, protocols, sample preparation or other tasks
including staged delivery and management of fluids.
[0019] A competition assay, such as enzyme multiplied immunoassay
technique (EMIT) may be conducted for qualitative and quantitative
analysis of components in a sample. Competition assays are
generally useful for detection of small molecules such as
metabolites. EMIT, for example, may be used for determination of
drugs and certain proteins. In a competition assay, an unlabeled
antigen and a labeling agent, for example a labeled antigen,
generally compete for a limited amount of specific antibody binding
sites. The unlabeled antigen may be associated with a target
analyte of the sample. During a competition assay, the unlabeled
antigen may be allowed to bind to an antibody. In some examples,
the antibody may be associated with (e.g. bound to) beads. In some
examples, the beads may be coated with the antibodies. It may be
advantageous to allow the unlabeled antigen to bind with the
antibody in the absence of the labeled antigen, so as to achieve
substantially complete binding of the unlabeled antigen in the
sample. During a competition assay, the labeled antigen may be
allowed to bind to unoccupied sites on the antibodies. In some
examples, the labeled antigen may be allowed to bind to
substantially all the unoccupied sites on the antibodies. In some
examples, the labeled antigen may compete with the target analyte
for sites on the antibodies. In this manner, the amount of labeled
antigen detected may be correlated to the amount of unlabeled
antigen in the sample, and therefore correlated to the amount of
target analyte in the sample. Thus, the amount of target analyte in
the sample may be quantified by detecting the amount of labeled
antigen bound to the antibodies. The amount of target analyte in
the sample may be inversely proportional to the amount of labeled
antigen in the sample. Thus, a strength of the signal detected may
be inversely proportional to the amount of the target analyte in
the sample.
[0020] FIG. 1 is a top down schematic illustration of an assay
device 100, according to one embodiment. The assay device 100 may
include features for conducting a competition assay, as will be
described below. In some examples, the assay device 100 may be
generally circular in shape. The assay device 100, including each
layer of the assay device 100, may be formed by known manufacturing
methods including, but not limited to, microfluidic manufacturing
techniques and semiconductor manufacturing techniques. Techniques
such as injection molding, cutting, or both, may be used. The assay
device 100 may be disposable in some examples. The assay device 100
may be portable in some examples.
[0021] The assay device 100 may include a control region 105 in
which a fluidic connection may be made between a first chamber 101
of the assay device 100 and a first portion 901 of a second chamber
102 of the assay device 100, as will be described below. The
control region 105 may be an area in one or more layers of the
assay device 100 in which an opening may be created. The assay
device 100 may include features in the control region 105 to
provide the fluidic connection. For example, pins may be placed in
the control region 105, as will be described below. In other
examples, wax or another sacrificial layer may be present in the
control region 105 that may be melted or otherwise removed to
create a fluidic connection. It may be advantageous to separate the
first chamber 101 and the second chamber 102 to perform certain
steps of a competitive assay, and then connect the first chamber
101 and the second chamber 102 to perform subsequent steps, as will
be described below. Additionally, the assay device 100 may be
manufactured so that the control region 105 in each layer
facilitates opening to create the fluid connection, for example
with perforations or a decreased thickness.
[0022] FIG. 2 is a top down exploded schematic illustration of an
assay device 100 including multiple layers, according to some
embodiments. In some examples, the assay device may include an
inlet layer 200, a first chamber layer 201, a second chamber layer
202, a bottom layer 203, and a control layer 204. It will be
understood that the assay device 100 may include additional layers
depending on a number of parameters of the assay, such as the
number of reagents involved, likelihood of cross-reactions, etc.
The additional layers may include additional chambers for loading a
number of samples and/or reagents. Furthermore, additional chambers
with a variety of features may be included in each layer.
Embodiments disclosed herein are merely examples, and one skilled
in the art will appreciate that chambers may be included in a
number of arrangements depending on the assay being conducted. The
inlet layer 200 may provide conduits for loading a sample and/or
reagents involved with the assay. The inlet layer 200 may be a
material formed by techniques such as but not limited to injection
molding, cutting, deposition, etching, or combinations thereof.
[0023] The inlet layer 200 may include one or more inlets for
loading of samples, reagents, or combinations thereof. The inlet
layer 200 may include a first chamber inlet 103 positioned adjacent
to a first chamber 101 of the assay device 100. The first chamber
inlet 103 may be an opening on an inlet layer 200 (see, e.g., FIGS.
2 and 4). The first chamber inlet 103 may provide a conduit whereby
a sample may be loaded into the first chamber 101. In some
examples, the first chamber inlet may also be used to load other
reagents, for example coated beads. In some examples, the first
chamber inlet may be sealed after the samples and other reagents
are loaded to prevent leakages.
[0024] The inlet layer 200 may include a second chamber inlet 104
positioned proximate to a second chamber 102 of the assay device
100. The second chamber inlet 102 may be an opening on the inlet
layer 200. The second chamber inlet 102 may be a conduit that
couples with one or more channels of one or more layers of the
assay device 100, whereby a passageway is created such that
reagents for conducting a competition assay may be loaded into the
second chamber 102. As will be described below, the second chamber
102 may be loaded with a density media and/or a labeling agent.
[0025] The inlet layer 200 may include a vent 106 to maintain a
relatively constant pressure while samples and/or reagents are
being loaded. It may be advantageous to maintain a relatively
constant pressure to prevent disturbances, such as bubbles forming,
when the first chamber 101 and/or the second chamber 102 are being
loaded or connected to one another. The vent 106 may be a conduit
that may couple with one or more channels of one or more layers of
the assay device 100, whereby a passageway is created such that
pressure gradients may be reduced.
[0026] The first chamber layer 201 of the assay device 100 may
include a first chamber 101, a first second chamber passage 205,
and a first vent passage 207. The first chamber layer 201 may be
positioned adjacent to the inlet layer 200. The first second
chamber passage 205 may couple with the second chamber inlet 104 to
provide a conduit for reagents to be loaded into the second chamber
102. The first vent passage 207 may couple with the vent 106 to
provide a conduit for air to pass such that pressure gradients
across the multiple layers of the assay device 100 may be
reduced.
[0027] The first chamber 101 may be a compartment, recess, or
another type of structure shaped to house a sample, beads coated
with antibodies, or combinations thereof. The first chamber 101 may
be coupled to the first chamber inlet 103 such that the reagents,
such as the sample and the coated beads, may be loaded into the
first chamber 101. In some examples, the coated beads may be
pre-loaded into the first chamber 101, for example the beads may be
loaded during manufacturing of the assay device 100. In this
manner, the assay device 100 may not require a user to load the
beads, thereby increasing the ease of use of the assay device 100.
In some examples, the first chamber 101 may be positioned adjacent
to a first portion 901 of the second chamber 102. The control
region 105 may be located anywhere that the first chamber 101 and
the second chamber 102 are adjacent to one another. In some
examples, the control region 105 may be located at a peripheral
region of the first chamber 101 that overlaps with a peripheral
region of the first portion 901 of the second chamber 102.
[0028] The first chamber layer 201 may be separated from the second
chamber layer 202 by a control layer 204. The control layer 204 may
be formed from a number of materials, such as a wax, a polymer, a
photoresponsive material, a chemoresponsive material, glass,
thermoplastics, or combinations thereof. The control layer 204 may
open at the control region 105 by, for example, being punctured or
by deforming in response to being exposed to a material or a
stimulus. A control layer 204 formed from a wax, for example
paraffin, may open responsive to a heat stimulus, which may cause
the wax to melt, thereby providing a fluid connection between the
first chamber 101 and the second chamber 102. As wax is typically
less dense than water, the molten wax may float on top of the
aqueous layer and not interfere with the assay. A control layer 204
formed from a polymer may open responsive to heating with an
infrared LED, which may cause the polymer to burn away, thereby
providing a fluid connection between the first chamber 101 and the
second chamber 102. A control layer 204 formed from a
photoresponsive material may open responsive to exposure to UV
light to provide a fluid connection between the first chamber 101
and the second chamber 102. In some examples, the photoresponsive
material may transform from a gel to a liquid responsive to the UV
light. A control layer 204 formed from a chemoresponsive material
may open responsive to a change in pH, which may cause some
materials to melt away and provide a fluid connection between the
first chamber 101 and the second chamber 102. A control layer 204
formed from a thermoplastic material, for example an acetate,
acrylate, polyethylene derivative, or combinations thereof, may
open responsive to a heat stimulus, which may cause the
thermoplastic to melt, thereby providing a fluidic connection
between the first chamber 101 and the second chamber 102.
[0029] The control layer 204 may have a thickness in the range of 2
.mu.m to 500 .mu.m, for example 150 .mu.m. In some examples, the
control layer 204 may be between 100-500 .mu.m thick, in some
examples 2-100 .mu.m thick, in some examples 100-200 .mu.m thick,
in some examples 200-300 .mu.m thick, in some examples 300-400
.mu.m thick, in some examples 400-500 .mu.m thick, in some examples
2-250 .mu.m thick, and in some examples 250-500 .mu.m thick. It may
be desirable to form the control layer 204 to be thick enough such
that it does not open on its own, but not so thick that it is
difficult to open. In some examples, a dye or an energy-absorbing
agent may be included in the control layer 204 to make it more
sensitized to the stimulus being used to open it. For example, when
using an infrared LED to open a control layer 204 formed from a
polymer, an infrared absorbing dye may be incorporated into the
control layer 204. Exposure to infrared light may cause the
infrared absorbing dye to rise in temperature, thereby facilitating
opening of the control layer 204. Similarly, a heat-sensitive dye
may be incorporated into the control layer 204 when using a heat
stimulus in order to facilitate opening of the control layer 204.
In some examples, the temperature may rise to a melting point of
the control layer 204.
[0030] The control layer 204 may include a second vent passage 208
that may be coupled to the first vent passage 207 to provide a
conduit whereby air may pass through such that pressure gradients
across the multiple layers of the assay device 100 may be reduced.
The control layer 204 may include a second second chamber passage
206 that may be coupled to the first second chamber passage 205 to
provide a conduit for reagents to be loaded into the second chamber
102. In some examples, multiple control layers may be provided to
provide fluidic connections between different sets of layers of the
assay device 100.
[0031] The second chamber layer 202 of the assay device 100 may
include a second chamber 102 coupled to one or more of the second
second chamber passage 206, the control region 105, and the second
vent passage 208. The second chamber layer 202 may be positioned
adjacent to the control layer 204. The second chamber 102 may be
loaded with reagents through the conduit created by the second
chamber inlet 104, the first second chamber passage 205 and the
second second chamber passage 206. The second chamber 102 may be
vented by the conduit provided by the vent 106, the first vent
passage 207, and the second vent passage 208.
[0032] The second chamber 102 may be a compartment, recess, or
another type of structure shaped to house a density media, a
labeling agent, or combinations thereof. The second chamber 102 may
be coupled to the second chamber inlet 104, the first second
chamber passage 205 and the second second chamber passage 206 such
that the reagents, such as the density media and the labeling
agent, may be loaded into the second chamber 102. In some examples,
the density media and the labeling agent may be pre-loaded into the
second chamber 102. For example the density media and the labeling
may be loaded during manufacturing of the assay device 100. In this
manner, the assay device 100 may not require a user to load the
density media or the labeling agent, thereby increasing the ease of
use of the assay device 100. The second chamber 102 may include a
first portion 901, a second portion 900, and a detection region
902. In some examples, the first portion of the second chamber 102
may be positioned adjacent to the first chamber 101. The control
region 105 may be positioned anywhere that the first chamber 101
and the second chamber 102 are adjacent to one another. In some
examples, the control region 105 may be located at a peripheral
region of the first chamber 101 that overlaps with a peripheral
region of the first portion 901 of the second chamber 102.
[0033] FIG. 3 is a bottom up schematic illustration of an assay
device 100, according to some embodiments. The assay device 100 may
be generally flat on a bottom side. In some examples, the assay
device 100 may include features on the bottom side for optical
encoding. The features may include patterns that may be used as a
rotary encoder rather than having an encoder on the motor 108. The
features may be tracked using an optical transceiver that may relay
positional information to the controller 1004. The features may be
formed by known manufacturing methods including, but not limited
to, microfluidic manufacturing techniques and semiconductor
manufacturing techniques. Techniques such as injection molding,
etching, cutting, or combinations thereof, may be used. In some
examples, etching may be used on the surface of the bottom layer
203 to create reflective and diffuse areas that may enable optical
encoding.
[0034] FIG. 4 is a bottom up exploded schematic illustration of an
assay device, according to some embodiments. The bottom layer 203
of the assay device 100 may be a substrate formed by techniques
such as injection molding, cutting, or both. The bottom layer 203
may provide a base for the assay device 100. In some examples, the
bottom layer may be positioned adjacent to the second chamber layer
202.
[0035] FIG. 5 is a cross-sectional schematic illustration of an
assay device 100, according to some embodiments. As shown in the
cross-sectional portion of FIG. 5, the first chamber inlet 103 may
be a conduit that passes through the inlet layer 200 and into the
first chamber 101. The bottom of the first chamber 101 may be
provided by the control layer 204. The second chamber inlet 104 may
be a conduit that passes through the inlet layer 200, the first
chamber layer 201 and the control layer 204 where it may reach the
second chamber 102. FIG. 5 shows an example where the second
chamber 102 extends further to the periphery of the assay device
100 compared to the first chamber 101. In some examples, the
peripheral end of the second chamber 102 may include a detection
region 902, as will be described below.
[0036] FIG. 6 is a cross-sectional schematic illustration of an
assay device 100 prior to opening a control layer 204, according to
some embodiments. Prior to opening the control layer 204, the first
chamber 101 may be fluidly disconnected from the second chamber
102. A pin 600 may be placed within or proximate to the assay
device 100, as will be described below. The first chamber may be
sized such that the contents of the first chamber 101 may be in the
laminar flow regime prior to opening the control layer 204. While
in the laminar flow regime, the contents of the first chamber 101
may not mix with each other, even during a time when the device may
be spinning to apply centrifugal force to the fluids contained
therein. The second chamber 102 may also be sized such that the
contents of the second chamber 102 may be in the laminar flow
regime prior to opening the control layer 204. While in the laminar
flow regime, the contents of the second chamber 101 may not mix
with each other, even during a time when the device may be spinning
to apply centrifugal force to the fluids contained therein.
[0037] FIG. 7 is a cross-sectional schematic illustration of an
assay device after opening a control layer, according to some
embodiments. In some examples, the assay device 100 may include
multiple assay sets, each of which includes a first chamber 101 and
a second chamber 102. In some examples, one or more pins 600 may be
used to open the control layer 204 at one or more control regions
105 of the assay device 100, thereby achieving a fluidic connection
of the chambers of each assay set. In some examples, the pins 600
may be positioned adjacent to the control regions 105 of the
control layer 204 such that they may be moved through the control
regions 105 responsive to a force or stimulus. In some examples,
the pins 600 may be incorporated into one of the layers, for
example the inlet layer 200 or the first chamber layer 201. In some
examples, the pins 600 may be formed of metal, and may be moved by
applying a magnetic field 701 across the assay device 100 by a
magnet 700, for example an electromagnet. The magnetic field 701
may apply a force to the pins 600 such that the pins 600 travel
through the control regions 105 and create an opening to provide a
fluid connection between each first chamber 101 and each first
portion 901 of the second chamber 102. In some examples, an
actuator may apply a mechanical force to move the pins 600 through
the control regions 105 to provide a fluidic connection between
each first chamber 101 and each first portion 901 of the second
chamber 102. In some examples, multiple pins 600 may be moved
simultaneously to open multiple control regions 105.
[0038] FIG. 7 shows an example where the control region 105 may be
at the peripheral end of the first chamber 101. However, it will be
understood that the control region 105 may be positioned at any
region in which the first chamber 101 and the first portion 901 of
the second chamber 102 are adjacent to one another. Furthermore,
the control region 105 may vary in size. For example, when the
entire first chamber 101 is adjacent to the first portion 901 of
the second chamber 102, the control region 105 may include the
entire first chamber 101. In this example, the control layer 204
may be opened to fully connect the first chamber 101 with the first
portion 901 of the second chamber 102. Thus, the control layer 204
may be at least partially opened to provide a fluid connection
between the first chamber 101 and the first portion 901 of the
second chamber 102. In some examples, there may be multiple control
regions that may be opened to provide fluidic connections between
two or more chambers. The multiple control regions may be
positioned on the same control layer or on multiple control
layers.
[0039] In some examples, the control layer 204 may be opened in
response to a stimulus, such as UV light, infrared light, or a pH
change. It may be advantageous to use a stimulus when the control
region 105 is an irregular shape or to ensure that the pins do not
obstruct pathways in the second chamber 102. UV light or infrared
light may be applied by known methods across the assay device 100,
whereby the light travels through the layers of the assay device
100 until it reaches the control layer 204. The control layer 204,
which may be formed from a photoresponsive material, may open in
response to exposure to the UV light or infrared light, thereby
providing a fluid connection between the first chamber 101 and the
first portion 901 of the second chamber 102. A pH change may be
initiated by introducing a base or an acid through the first
chamber inlet 101. The base or acid may change the pH of the
contents of the first chamber 101, causing the control layer 204 to
open, thereby providing a fluid connection between the first
chamber 101 and the first portion 901 of the second chamber
102.
[0040] Upon opening the control layer 204, the contents of the
first chamber 101 and the first portion 901 of the second chamber
102 may mix together. The device may be agitated and/or spun to
facilitate the mixing. The increase in the channel (or chamber)
height from combining the first chamber 101 and the first portion
901 of the second chamber 102 by opening or deforming the control
layer may result in the overall combined fluidic feature moving
from the laminar flow regime to a turbulent flow regime.
Accordingly, the contents of the first chamber 101 and the first
portion 901 of the second chamber 102 may mix responsive to the
turbulent flow in the first portion of the second chamber, which
may be generated by moving and/or agitating the device 100, for
example. In some examples, the assay device 100 may be moved to
achieve mixing. A motor or manual force may be used to move the
assay device 100. The channel height of the second portion 900 of
the second chamber 102 may remain constant after opening the
control layer 204. Thus, the contents of the second portion 900 may
stay in the laminar flow regime. Therefore, the mixing may occur in
the first portion 901 of the second chamber 102 without mixing
contents of the second portion 900 of the second chamber 102.
[0041] FIG. 8 is a flowchart depicting a method for performing an
assay, according to embodiments described herein. Operation 800
involves loading a sample into a first chamber (e.g. 101 of FIG. 2)
of an assay device, wherein the first chamber is loaded with beads
coated with antibodies. Operation 802 involves incubating the
sample and beads to allow formation of complexes of beads and the
target analyte when present in the sample. In some examples, the
incubation time may be in the range of about 0.5 minutes to about 6
hours. Operation 804 involves opening at least a portion of a
control layer (e.g. 204 of FIG. 2) dividing the first chamber from
a second chamber (e.g. 102 of FIG. 2) to provide a fluidic
connection between the first chamber and a first portion (e.g. 901
of FIG. 9) of the second chamber, wherein the second chamber
contains a labeling agent in the first portion of the second
chamber and a density media in a second portion (e.g. 900 of FIG.
9) of the second chamber, wherein the labeling agent is configured
to compete for sites on the antibodies with the target analyte. In
some examples, the second chamber may be vented after opening at
least a portion of the control layer through a vent (e.g. 106 of
FIG. 1) coupled to the second chamber. In some examples, the beads
may be transported from the first chamber to the second chamber by
a pressure gradient after providing the fluidic connection. In some
examples, the plurality of beads and the labeling agent may be
mixed after opening at least a portion of the control layer.
Operation 806 involves allowing the labeling agent to bind to
unoccupied sites on the antibodies in the first portion of the
second chamber. Operation 808 involves transporting the beads
through the density media, wherein the density media has a density
lower than a density of the beads.
[0042] Operation 810 involves detecting signal from labeling agents
bound to the antibodies. The measurement may be performed using the
detection unit. In some examples, the measurement may be performed
during the predetermined interval where the assay device 100 is
stopped, for example by stopping a stepper motor or engaging a
stopping mechanism. For example, the measurement may detect
fluorescence present in the detection region, which may allow
identification of analyte including, but not limited to
biomolecules such as proteins, nucleic acids, or combinations
thereof. The method of FIG. 8 may be implemented using any of the
structures shown in FIGS. 1-7 and FIGS. 9-10 in some examples.
[0043] FIG. 9 is a top down exploded schematic illustration of the
first chamber layer 201, the control layer 204, and the second
chamber layer 202, according to some embodiments. The first portion
901 of the second chamber 102 may contain a first reagent, for
example the labeling agent. The labeling agent may be any suitable
labeling agent for binding to the antibodies and providing a
detection signal. Examples include chemical dyes and/or nucleic
acid dyes. Fluorescent labels including the aforementioned chemical
dyes may provide an optical detection signal, however colorimetric
or radioactive tags may also be used. The first portion 901 may be
a compartment, recess, or another type of structure. The first
portion 901 may be coupled to the second portion 900 of the second
chamber 102. Prior to opening the control layer 204, the first
portion 901 may be sized for laminar flow. While in the laminar
flow regime, the first reagent may be substantially static such
that little mixing occurs between the first reagent and a second
reagent in the second portion 900. In some examples, the end of the
first portion 901 closest to the second portion 900 may be
relatively narrow so as to further inhibit mixing between the first
reagent and the second reagent.
[0044] The second portion 900 of the second chamber 102 may contain
the second reagent, for example the density media. The second
portion 900 may be a compartment, recess, or another type of
structure. The second portion 900 may be coupled to the first
portion 901. The second portion 900 may be sized for laminar flow.
While in the laminar flow regime, the second reagent may be
substantially static such that little mixing occurs between the
first reagent and the second reagent. In some examples, the end of
the second portion 900 closest to the first portion 901 may be
relatively narrow so as to further inhibit mixing between the first
reagent and the second reagent. In some examples, the second
portion 900 of the second chamber 102 may stay in the laminar flow
regime after a fluidic connection is established between the first
chamber 101 and the first portion 901 of the second chamber 102.
Thus, the density media loaded in the second portion 900 may not
mix with the complexes formed between the coated antibodies, target
antigen, and the labeling agent until a centrifugal force is
applied, as will be described below.
[0045] The density media may have a density greater than the
sample, but less than that of the beads. For example, a blood
sample may have a density less than or equal to 1.095 g/cm.sup.3,
and beads formed from silica may have a density of about 2.05
g/cm.sup.3. Accordingly, the density may have a density of between
1.095 g/cm.sup.3 and 2.05 g/cm.sub.3. In some examples, the density
media may have a density of 1.11 g/cm.sup.3. The density media may
include, for example, a salt solution containing a suspension of
silica particles which may be coated with a biocompatible coating.
An example of a suitable density media is Percoll.TM., available
from GE Lifesciences.
[0046] A detection region 902 may be positioned along a known path
to align with a detection unit 1002. In some examples, the known
path may be defined by a radius from the center of a circular assay
device 100. In some examples, the detection region 902 may be
coupled to the first portion 901 of the second chamber 102. The
detection region 902 may be a compartment, recess or another type
of structure shaped to concentrate a component to be detected by
the detection unit 1002. In some examples, the detection region 902
may be shaped to accommodate beads that may get pelleted through
the density media responsive to a centrifugal force applied by a
motor, as will be described below.
[0047] FIG. 10 is a schematic illustration of a system for
conducting an assay, according to some embodiments. The system may
include the assay device 100, a motor 1000, a detection unit 1002,
and a controller 1004. The motor 1000 may move the assay device 100
such that multiple detection regions may move along a known path.
In some examples, the path may be circular. In some examples, the
path may be linear. The motor 1000 may be a DC motor coupled to the
assay platform 100, such as a stepper motor. In some examples,
other types of motors, such as solenoid motors, servo motors, or
combinations thereof may be used in conjunction with a stopping
mechanism. The stopping mechanism may allow the assay device 100 to
be moved in a known manner such that the detection regions 902
present on the assay device 100 may sequentially align with the
detection unit 1002.
[0048] In some examples, the motor 1000 may apply a centrifugal
force to an assay device 100 mounted in the system. Centrifugal
force may be used to separate one or more components of one or more
samples positioned within the connected first chamber and second
chamber of the assay platform 100. For example, components of
biological and clinical samples may need to be separated to
facilitate measurement (e.g. detection and/or quantification) of an
analyte. Applying a centrifugal force may achieve a separation of
one or more components of the biological or clinical samples placed
within one or more of the connected first chambers and second
chambers of the assay platform 100. In some examples, a fluid
sample positioned within one or more of the connected first
chambers and second chambers may include a plurality of beads
having complexes formed thereon, in which individual ones of the
complexes include a target analyte and a labeling agent bound to
the beads in cooperation with the target analyte. The fluid sample
may also include free labeling agent that may be unbound. The motor
1000 may apply a centrifugal force to the fluid sample, whereby the
beads in the fluid sample may be transported responsive to the
centrifugal force through a density media to pellet out at the
detection region 902. Free labeling agent may have insufficient
density to be transported through the density media and may not be
present at the detection region 902. It should be understood that
the motor 1000 may be configured to move the assay device 100 to
effect a separation of a variety of different types of assays
responsive to a centrifugal force. Examples of assays usable with
examples of the present invention are described in the applications
incorporated by reference above.
[0049] The detection unit 1002 may perform measurements to detect
and/or quantify an analyte in any or all of the detection regions
902 present on the assay device 100. The detection unit 1002 may,
for example, include an optical light sensor for performing optical
measurements, such as fluorescence, luminescence, and/or relative
light absorption. In some examples, other sensors (e.g. electrical
sensors) may be included additionally or instead in the detection
unit 1002 to support other detection methodologies. The detection
unit 1002 may be mounted in a system in proximity to a mount for
holding the assay device 100. Generally, the detection unit 1002 is
positioned in a system such that a detection region 902 is aligned
with the detection unit 1002 such that detection unit 1002 is
positioned to take a measurement from the detection region 902, or
to move in a known manner to the detection region 902. A mount may
be provided in a system for receiving the assay device 100 which
may generally be inserted into and removed from the system. The
detection unit 1002 may accordingly be positioned in a known manner
relative to the mount to facilitate alignment between detection
regions on devices that may be placed on the mount and the
detection unit 1002.
[0050] In some examples, the controller 1004 may be communicatively
coupled (e.g. electrically) to the motor 1000. The controller 1004
may be an electronic device, for example a computing device, that
may transmit control signals at predefined times and/or predefined
intervals to recipient devices. Additionally, the controller 1004
may store timing information using a timing device, for example a
timer integrated circuit. The control signals may be transmitted
using an implementation of a protocol recognizable by the recipient
devices. The recipient device may include the detection unit 1002
and/or the motor 1000. The controller 1004 may receive user input
that defines parameters of the measurement system, such as distance
between each of the detection regions 902, rotational speed, etc.
The controller 1004 may provide control signals to the motor 1000
to sequentially measure each detection region 902 of the assay
device 100. In some examples, the controller may communicate with
an optical transceiver to receive positional information. In some
examples, the controller 1004 may stop the motor 1000 for a
predetermined period of time at each detection region 902 to make a
measurement. It may be advantageous to take a measurement while the
assay device 100 is not moving in order to increase the integration
time for light (or other signal) collection, which may increase the
signal to noise ratio and hence sensitivity. It may be advantageous
to stop the motor 1000 for a predetermined period of time to reduce
electrical noise originating from the active motor during analysis
by the detection unit 1002. After a predetermined time, the
controller 1004 may again provide control signals to start the
motor 1000 to move the assay device 100 such that the next
detection region 902 may be in alignment with the detection unit
1002.
[0051] In some examples, the controller 1004 may receive an
indication that a sample was received in the first chamber 101. The
indication may be provided by a sensor positioned inside or
proximate to the first chamber 101. After receiving the indication,
the controller 1004 may allow an incubation period to take place,
after which it may provide a control signal to open at least a
portion of the control layer 204. In some examples, the control
signal may be provided to an actuator, which may apply a mechanical
force to create an opening in the control layer 204. For example,
the mechanical force may be applied to one or more pins, whereby
the pins 600 puncture the control layer 204 to provide a fluidic
connection between the first chamber 101 and the first portion 901
of the second chamber 102. In some examples, the control signal may
be provided to a magnet 700 to provide a magnetic force 701 to move
metal pins 600 through the control layer 204. In some examples, the
control signal 204 may be provided to known devices to deliver
ultraviolet light, infrared light, an acid or base, or combinations
thereof.
[0052] In some examples, the controller 1004 may provide a control
signal to the motor 1000 to move the assay device 100 to achieve
mixing. Mixing may be desired at a number of stages of a
competition assay. In some examples, mixing may be desired after
loading the sample into the first chamber 101 in order to
facilitate binding of the sample to the antibodies in the first
chamber 101. As described above, the antibodies may be coated on to
beads. In some examples, mixing may be desired after providing a
fluidic connection between the first chamber 101 and the first
portion 901 of the second chamber 102 in order to facilitate
binding of the labeling agent to unoccupied sites on the
antibodies. In some examples, the motor 1000 may rotate the assay
device 100 back and forth to achieve mixing in the first portion
901 of the second chamber 102.
[0053] In some examples, the controller 1004 may provide a control
signal to the motor 1000 to move the assay device 100 to apply a
centrifugal force. A centrifugal force may be achieved by spinning
the assay device 100 using the motor 1000. As described above, the
centrifugal force may be used to separate the contents of one or
more assay sets of the assay device 100. In some examples, beads
may be transported through the density media in response to the
centrifugal force applied by the motor 1000. The beads may be
transported to the detection region 902 for measurement by the
detection unit 1002.
[0054] The assay device 100 may be used to perform a variety of
tasks involving staged delivery and management of fluids. In some
examples, the assay device 100 may be used to conduct a bead-based
nucleic acid assay, such as a PCR assay or a hybridization-based
assay. The nucleic acid assay may utilize any of molecular beacons
and Taqman chemistry. In addition, the assay device may be used to
conduct immunoassays, such as a sandwich assay. Further, the assay
device 100 may be used to conduct an affinity assay using aptamers,
peptides, PNAs, SCFVs, or combinations thereof. In addition, the
assay device 100 may be used to conduct an assay utilizing
enzymatic activity. The enzymatic activity may provide an
amplification of the detection signal. Further, the assay device
100 may be used to perform sample preparation. The sample
preparation technique may include a buffer exchange, for example
transferring beads from a clinical or environmental sample to an
assay buffer. In some examples, the buffer exchange may change the
pH or the ionic strength of the sample. In some examples, the
buffer exchange may stabilize the sample by using a second buffer
including stabilization reagents. The stabilized sample may be
archived and retrieved from the assay device 100.
[0055] From the foregoing it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
* * * * *