U.S. patent application number 16/015276 was filed with the patent office on 2019-05-30 for methods and compositions for detecting multiple analytes with a single signal.
The applicant listed for this patent is Invisible Sentinel, Inc.. Invention is credited to Ashley Shaniece Brown, Martin Patrick Keough, Louis Leong, Nicholas Siciliano.
Application Number | 20190162719 16/015276 |
Document ID | / |
Family ID | 49114453 |
Filed Date | 2019-05-30 |
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United States Patent
Application |
20190162719 |
Kind Code |
A1 |
Siciliano; Nicholas ; et
al. |
May 30, 2019 |
Methods And Compositions For Detecting Multiple Analytes With A
Single Signal
Abstract
Compositions, methods, and devices for the detection of multiple
analytes with a single signal are provided.
Inventors: |
Siciliano; Nicholas; (Cherry
Hill, NJ) ; Leong; Louis; (Junction City, OR)
; Keough; Martin Patrick; (Lansdowne, PA) ; Brown;
Ashley Shaniece; (Philadelphia, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Invisible Sentinel, Inc. |
Philadelphia |
PA |
US |
|
|
Family ID: |
49114453 |
Appl. No.: |
16/015276 |
Filed: |
June 22, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15161690 |
May 23, 2016 |
10018626 |
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16015276 |
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13789002 |
Mar 7, 2013 |
9347938 |
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15161690 |
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61608774 |
Mar 9, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/54306 20130101;
G01N 33/553 20130101; G01N 33/54366 20130101; B01L 2300/0627
20130101; C12Q 1/686 20130101; B01L 2300/069 20130101; C12Q 1/6804
20130101; G01N 33/543 20130101; B01L 3/5023 20130101; G01N 33/54353
20130101 |
International
Class: |
G01N 33/543 20060101
G01N033/543; C12Q 1/686 20060101 C12Q001/686; C12Q 1/6804 20060101
C12Q001/6804; B01L 3/00 20060101 B01L003/00 |
Claims
1-46. (canceled)
47. A complex comprising: a first capture reagent affixed to a
solid support; a first analyte of interest comprising a first
interaction unit and a second interaction unit; a second analyte of
interest comprising a first interaction unit and a second
interaction unit; a bridge unit; and a signal detection unit,
wherein the first capture reagent binds to the first interaction
unit of the first analyte of interest, wherein the bridge unit
comprises one or more capture reagents that independently bind to
the second interaction unit of the first analyte and the first
interaction unit of the second analyte of interest; and the signal
detection unit comprises a capture reagent that binds to the second
interaction unit of the second analyte of interest, wherein the
first analyte of interest and the second analyte of interest are E.
coli analytes.
48. The complex of claim 47, wherein the first analyte of interest
and the second analyte of interest are different E. coli
analytes.
49. The complex of claim 47, wherein the first analyte of interest
and the second analyte of interest are amplicons.
50. The complex of claim 47, wherein the first analyte of interest
and the second analyte of interest are independently amplicons of
Shiga 1 toxin, Shiga 2 toxin, or eae.
51. The complex of claim 47, wherein the first analyte of interest
and the second analyte of interest are independently amplicons of
Shiga 1 toxin or eae.
52. The complex of claim 47, wherein the first analyte of interest
and the second analyte of interest are independently amplicons of
Shiga 2 toxin or eae.
53. The complex of claim 47, wherein the signal detection unit
emits a detectable signal.
54. The complex of claim 47, wherein the signal detection unit
comprises colloidal gold, a radioactive tag, a fluorescent tag, or
a chemiluminescent substrate.
55. The complex of claim 47, wherein the signal detection unit
comprises a detectable nanocrystal, a functionalized nanoparticle,
an up-converting nanoparticle, a cadmium selenide/cadmium sulfide
fusion nanoparticle, a quantum dot, a near-infrared fluorophore, a
lanthanide cluster, a phthalocyanine, a light emitting-diode.
56. A complex comprising: a first capture reagent affixed to a
solid support; a first analyte of interest comprising a first
interaction unit and a second interaction unit; a second analyte of
interest comprising a first interaction unit and a second
interaction unit; a third analyte of interest comprising a first
interaction unit and a second interaction unit; a first bridge
unit; a second bridge unit; and a signal detection unit comprising
a second capture reagent that binds to the second interaction unit
of the third analyte of interest, wherein the first capture reagent
binds to the first interaction unit of the first analyte of
interest; wherein the first bridge unit comprises one or more
capture reagents that independently bind to the second interaction
unit of the first analyte of interest and the first interaction
unit of the second analyte of interest; wherein the second bridge
unit comprises one or more capture reagents that independently bind
to the second interaction unit of the second analyte of interest
and the first interaction unit of the third analyte of interest;
and wherein the first analyte of interest, the second analyte of
interest, and the third analyte of interest are E. coli
analytes.
57. The complex of claim 56, wherein the first analyte of interest,
the second analyte of interest, and the third analyte of interest
are different E. coli analytes.
58. The complex of claim 56, wherein the first analyte of interest,
the second analyte of interest, and the third analyte of interest
are amplicons.
59. The complex of claim 56, wherein the first analyte of interest,
the second analyte of interest, and the third analyte of interest
are independently amplicons of Shiga 1 toxin, Shiga 2 toxin, or
eae.
60. The complex of claim 56, wherein the first analyte of interest
and the second analyte of interest are independently amplicons of
Shiga 1 toxin, Shiga 2 toxin, or eae
61. The complex of claim 56, wherein the signal detection unit
emits a detectable signal.
62. The complex of claim 56, wherein the signal detection unit
comprises colloidal gold, a radioactive tag, a fluorescent tag, or
a chemiluminescent substrate.
63. A complex comprising: a first analyte of interest comprising a
first interaction unit and a second interaction unit; a second
analyte of interest comprising a first interaction unit and a
second interaction unit; a bridge unit; and a signal detection
unit, wherein the bridge unit comprises one or more capture
reagents that independently bind to the second interaction unit of
the first analyte and the first interaction unit of the second
analyte of interest; and the signal detection unit comprises a
first capture reagent that binds to the second interaction unit of
the second analyte of interest, wherein the first analyte of
interest and the second analyte of interest are amplicons.
64. The complex of claim 63, further comprising a second capture
reagent affixed to a solid support; wherein the second capture
reagent binds to the first interaction unit of the first analyte of
interest.
65. A complex comprising: a first analyte of interest comprising a
first interaction unit and a second interaction unit; a second
analyte of interest comprising a first interaction unit and a
second interaction unit; a third analyte of interest comprising a
first interaction unit and a second interaction unit; a first
bridge unit; a second bridge unit; and a signal detection unit
comprising a first capture reagent that binds to the second
interaction unit of the third analyte of interest, wherein the
first bridge unit comprises one or more capture reagents that
independently bind to the second interaction unit of the first
analyte of interest and the first interaction unit of the second
analyte of interest; wherein the second bridge unit comprises one
or more capture reagents that independently bind to the second
interaction unit of the second analyte of interest and the first
interaction unit of the third analyte of interest; and wherein the
first analyte of interest, the second analyte of interest, and the
third analyte of interest are amplicons.
66. The complex of claim 65, further comprising a second capture
reagent affixed to a solid support; wherein the second capture
reagent binds to the first interaction unit of the first analyte of
interest.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 13/789,002, filed Mar. 7, 2013, which claims priority to U.S.
Provisional Application No. 61/608,774, filed Mar. 9, 2012, each of
which is hereby incorporated by reference in its entirety.
FIELD OF INVENTION
[0002] Embodiments are directed to, in part, the detection of
multiple analytes with a single signal.
BACKGROUND OF INVENTION
[0003] The detection of multiple analytes often requires the use of
multiple signals or multiple reactions, spots, or wells to
determine if a sample has multiple analytes. This can complicate
interpretation and, in cases where an adulterant is classified as
having two or more detectable characteristics, can make
identification challenging for the end-user. Thus, to simplify and
provide a consolidated qualitative report to the end-user, there is
a need for methods and compositions that enable the detection of
multiple analytes in a sample with a single signal. The present
invention satisfies this need and others.
SUMMARY OF THE INVENTION
[0004] The present invention provides methods of concurrently
detecting a first analyte and a second analyte comprising:
contacting a solid support with a first analyte, a second analyte,
a bridge unit comprising a second capture reagent, and a signal
detection unit comprising a third capture reagent; and detecting
the presence or absence of the signal detection unit which
indicates the presence or absence of the first analyte and second
analyte concurrently, wherein a first capture reagent is affixed to
the solid support; the first analyte comprises a first interaction
unit that binds to the first capture reagent and a second
interaction unit that binds to the bridge unit; and the second
analyte comprises a first interaction unit that binds the bridge
unit and a second interaction unit that binds to the signal
detection unit.
[0005] The present invention also provides methods of concurrently
detecting a first analyte, a second analyte, and a third analyte
with a single signal comprising: contacting the first, second, and
third analytes with a solid support, a first bridge unit, a second
bridge unit, and a signal detection unit; and detecting the
presence of the signal detection unit which indicates the presence
of the first, second, and third analytes concurrently with a single
signal, wherein: the first analyte comprises a first interaction
unit and a second interaction unit; the second analyte comprises a
first interaction unit and a second interaction unit; the third
analyte comprises a first interaction unit and a second interaction
unit; the solid support comprises a first capture reagent that
binds to the first interaction unit of the first analyte; the first
bridge unit binds to the second interaction unit of the first
analyte and the first interaction unit of the second analyte; the
second bridge unit binds to the second interaction unit of the
second analyte and the first interaction unit of the third analyte;
and the signal detection unit binds to the second interaction unit
of the third analyte. The interaction units can be different from
one another on each of the analytes.
[0006] In some embodiments, methods of concurrently detecting a
first analyte and a second analyte are provided, the method
comprising: contacting a solid support with a first analyte of
interest, a second analyte of interest, a bridge unit comprising a
second capture reagent, and a signal detection unit comprising a
third capture reagent; and detecting the presence or absence of the
signal detection unit which indicates the presence or absence of
the first analyte of interest and second analyte of interest
concurrently, wherein: a first capture reagent is affixed to the
solid support; the first analyte of interest comprises a first
interaction unit that binds to the first capture reagent and a
second interaction unit that binds to the bridge unit; and the
second analyte of interest comprises a first interaction unit that
binds the bridge unit; a signal detection unit that binds to the
second analyte, to the second analyte's first interaction unit or a
second interaction unit, to a component of the first and second
analyte complex or bridge unit that that is only present when the
complex contains the first and second analyte.
[0007] Embodiments described herein also provide complexes
comprising a solid support, a first analyte, a second analyte, a
bridge unit, and a signal detection unit wherein each member of the
complex binds to each other directly or indirectly.
[0008] Embodiments described herein also provide complexes
comprising a solid support, a first analyte, a second analyte, a
third analyte, a first bridge unit, a second bridge unit, and a
signal detection unit, wherein the solid support, first analyte,
second analyte, third analyte, first bridge unit, second bridge
unit, and signal detection unit are bound to each other directly or
indirectly.
[0009] Methods of concurrently detecting a first analyte of
interest and a second analyte of interest are provided herein. In
some embodiments, the method comprises contacting a solid support
with a first analyte of interest, a second analyte of interest, a
bridge unit comprising a second capture reagent, and a signal
detection unit comprising a third capture reagent; and detecting
the presence or absence of the signal detection unit which
indicates the presence or absence of the first analyte of interest
and second analyte of interest concurrently, wherein a first
capture reagent is affixed to the solid support; the first analyte
of interest comprises a first interaction unit that binds to the
first capture reagent and a second interaction unit that binds to
the bridge unit; and the second analyte of interest comprises a
first interaction unit and a second interaction unit, wherein the
first interaction unit binds the bridge unit; a signal detection
unit that binds to: i) the second analyte, ii) to the second
analyte's first interaction unit or second interaction unit, iii)
to a component of the first and second analyte complex, or iv)a
component of an analyte-bridge complex that is only present when
the complex contains the first and second analytes.
[0010] In some embodiments, the first and second interaction unit
of the first analyte of interest and the first and second
interaction unit of the second analyte of interest are each,
independently, a heterologous interaction unit. In some
embodiments, the second interaction unit of the first analyte of
interest and the first interaction unit of the second analyte of
interest comprise the same heterologous interaction unit. In some
embodiments, the second interaction unit of the first analyte of
interest and the first interaction unit of the second analyte of
interest comprise different heterologous interaction units. In some
embodiments, the first interaction unit of the first analyte of
interest and the second interaction unit of the second analyte of
interest comprise the same heterologous interaction unit. In some
embodiments, the first interaction unit of the first analyte of
interest and the second interaction unit of the second analyte of
interest comprise different heterologous interaction units.
[0011] Methods of concurrently detecting a first analyte of
interest, a second analyte of interest, and a third analyte of
interest with a single signal are provided. In some embodiments,
the method comprises contacting the first, second, and third
analytes of interest with a solid support, a first bridge unit, a
second bridge unit, and a signal detection unit; and detecting the
presence of the signal detection unit which indicates the presence
of the first, second, and third analytes of interest concurrently
with a single signal, wherein: the first analyte of interest
comprises a first interaction unit and a second interaction unit;
the second analyte of interest comprises a first interaction unit
and a second interaction unit; the third analyte of interest
comprises a first interaction unit and a fifth interaction unit;
the solid support comprises a first capture reagent that binds to
the first interaction unit of the first analyte of interest; the
first bridge unit binds to the second interaction unit of the first
analyte of interest and the first interaction unit of the second
analyte of interest; the second bridge unit binds to the second
interaction unit of the second analyte of interest and the first
interaction unit of the third analyte of interest; and the signal
detection unit binds to: i) the third analyte, ii) to the third
analyte's first interaction unit or second interaction unit, iii)
to a component of the first, second, or third analyte complex, or
iv) a component of an analyte-bridge complex that is only present
when the complex contains the first, second, and third
analytes.
[0012] In some embodiment, the bridge units described herein are
multivalent capture reagents. In some embodiments, the multivalent
capture reagent is an immunoglobulin. In some embodiments, the
immunoglobulin is IgM. The bridge unit can also be biotin.
[0013] Methods of concurrently detecting a plurality of analytes
with a single signal with a device are provided. In some
embodiments, the method comprises a) contacting a device for
detecting a plurality of analytes with a single signal with one or
more samples comprising a plurality of analytes, wherein the device
comprises: a housing comprising: an inlet opening in fluid contact
with a conjugate pad; a force member; a slidable locking member
contacting the force member; an attachment member contacting the
force member; a sliding button contacting the attachment member;
and a detection membrane system comprising the conjugate pad, a
test membrane, and an absorbent member, at least a portion of the
conjugate pad, test membrane, and absorbent member are
substantially parallel to each other, the force member contacts the
detection membrane system and is capable of applying pressure
substantially perpendicular to the detection membrane system, the
sliding button moves the slidable locking member, the conjugate pad
comprises a signal detection unit comprising a third capture
reagent; the test membrane comprises a first capture reagent
affixed to the test membrane; wherein the one or more samples
comprises a first analyte of interest, a second analyte of
interest, and a bridge unit comprising a second capture reagent,
wherein the first analyte of interest comprises a first interaction
unit that binds to the first capture reagent and a second
interaction unit that binds to the bridge unit, and the second
analyte of interest comprises a first interaction unit that binds
the bridge unit and a second interaction unit; wherein the signal
detection unit comprising the third capture reagent binds to: i)
the second analyte, ii) to the second analyte's first interaction
unit or second interaction unit, iii) to a component of the first
and second analyte complex, or iv) a component of an analyte-bridge
complex that is only present when the complex contains the first
and second analytes; and b) detecting the presence or absence of
the signal detection unit which indicates the presence or absence
of the first analyte of interest and second analyte of interest
concurrently.
[0014] In some embodiments, the method comprises moving the
conjugate pad after a portion of the one or more samples has
contacted and flowed through the conjugate pad, thereby exposing at
least a portion of the test membrane for detection of the signal
detection unit to indicate the presence or absence of the plurality
of analytes with a single signal. In some embodiments, the
conjugate pad is moved by moving the slidable locking member. In
some embodiments, the first and second analyte are amplicons. In
some embodiments, the first and second analytes are PCR reaction
products. In some embodiments, the first analyte's first
interaction unit is a digoxigenin label. In some embodiments, the
first analyte's second interaction unit is a rhodamine label. In
some embodiments, the second analyte's first interaction unit is a
rhodamine label. In some embodiments, the second analyte's second
interaction unit is a fluorescein label. In some embodiments, the
third capture reagent binds to the second analyte's second
interaction unit. In some embodiments, the third capture reagent is
a biotinylated capture reagent. In some embodiments, the signal
interaction unit is coated with streptavidin. In some embodiments,
the signal interaction unit is streptavidin coated colloidal gold.
In some embodiments, the first and second analytes are nucleic acid
amplification products, wherein: the first analyte comprises a
digoxigenin label and a rhodamine label; the second analyte
comprises a rhodamine label and a fluorescein label; the first
capture reagent is an anti-digoxigenin label antibody; the second
capture reagent is an anti-rhodamine label antibody; the third
capture reagent is a biotinylated anti-fluorescein label antibody;
and the signal interaction unit is streptavidin coated colloidal
gold.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 illustrates, among other aspects, the representative
detection of two analytes with a single signal.
[0016] FIG. 2 illustrates, among other aspects, the representative
detection of three analytes with a single signal.
[0017] FIG. 3 illustrates, among other aspects, two amplification
products being detected with colloidal gold.
[0018] FIG. 4 illustrates, among other aspects, a multi-component
bridging unit
[0019] FIG. 5 illustrates, among other aspects, the representative
detection of two analytes with a single signal using a
multi-component bridging unit.
[0020] FIG. 6 illustrates, among other aspects, the signal
detection unit binding to a component of the bridging unit that is
only present when the plurality of analytes is present in the
complex.
[0021] FIG. 7 illustrates, among other aspects, a non-limiting
workflow for detecting a plurality of analytes with a single
signal.
[0022] FIG. 8 depicts a perspective view of a representative device
according to some embodiments of the present invention.
[0023] FIG. 9 depicts some components of a representative device
according to some embodiments of the present invention.
[0024] FIG. 10 depicts some components of a representative device
according to some embodiments of the present invention.
[0025] FIG. 11 depicts some components of a representative device
according to some embodiments of the present invention.
[0026] FIG. 12 depicts some components of a representative device
in various positions according to some embodiments of the present
invention.
[0027] FIG. 13: Depicts a lateral view of some components of a
representative device according to some embodiments of the present
invention.
[0028] FIG. 14 depicts a lateral view of some components of a
representative device according to some embodiments of the present
invention.
[0029] FIG. 15A depicts a lateral view of some components of a
representative device according to some embodiments of the present
invention.
[0030] FIG. 15B depicts a view of some components, such as but not
limited to, a non-flexible attachment member, of a representative
device according to some embodiments of the present invention.
[0031] FIG. 15C depicts a perspective view of a representative
device according to some embodiments of the present invention.
[0032] FIG. 15D depicts a perspective view of a representative
device according to some embodiments of the present invention.
[0033] FIG. 16 depicts a flexible attachment member attached to a
conjugate pad.
[0034] FIG. 17 depicts membranes in a representative housing
member.
[0035] FIG. 18 depicts a side view and a top view of a
representative device according to some embodiments of the present
invention.
[0036] FIG. 19 depicts one type of analyte detection membrane
system for a representative device according to some embodiments of
the present invention.
[0037] FIG. 20 depicts one type of analyte detection membrane
system for a representative device according to some embodiments of
the present invention.
[0038] FIG. 21 depicts one type of analyte detection membrane
system for a representative device according to some embodiments of
the present invention.
[0039] FIG. 22 depicts one type of analyte detection membrane
system for a representative device according to some embodiments of
the present invention.
[0040] FIG. 23 depicts representative force members for a
representative device according to some embodiments of the present
invention.
[0041] FIGS. 24A-D depict a representative device according to some
embodiments of the present invention.
[0042] FIGS. 25A-C depict a representative device according to some
embodiments of the present invention.
[0043] FIGS. 26 depicts representative devices according to some
embodiments of the present invention.
[0044] FIGS. 27A-B depict a view of a representative device
according to some embodiments of the present invention.
[0045] FIG. 28 depicts an underneath view of a representative
device according to some embodiments of the present invention.
[0046] FIG. 29 depicts an exploded view of a representative device
according to some embodiments of the present invention.
[0047] FIG. 30 depicts an interior view of a representative device
according to some embodiments of the present invention.
[0048] FIGS. 31A-B depict a cross-sectional view of a
representative device according to some embodiments of the present
invention.
[0049] FIG. 32 depicts an exploded view of a representative device
according to some embodiments of the present invention.
[0050] FIG. 33 depicts an interior view of a representative device
according to some embodiments of the present invention.
[0051] FIG. 34 depicts a cross-sectional view of a representative
device according to some embodiments of the present invention.
[0052] FIG. 35 depicts a representative movable locking member
according to some embodiments of the present invention.
[0053] FIG. 36 depicts a representative housing according to some
embodiments of the present invention.
[0054] FIG. 37 depicts a representative housing according to some
embodiments of the present invention.
[0055] FIG. 38A depicts a representative device according to some
embodiments of the present invention.
[0056] FIG. 38B depicts a representative device according to some
embodiments of the present invention.
[0057] FIG. 39 depicts an enlarged view of a representative device
according to some embodiments of the present invention.
[0058] FIG. 40 depicts an exploded view of a cartridge and analyte
detection membrane system according to some embodiments of the
present invention.
[0059] FIG. 41 depicts a representative device according to some
embodiments of the present invention.
[0060] FIG. 42 depicts a representative device according to some
embodiments of the present invention.
[0061] FIGS. 43A-C depict a representative device according to some
embodiments of the present invention.
[0062] FIG. 44 depicts an exploded view of a representative device
according to some embodiments of the present invention.
[0063] FIG. 45 depicts an exploded view of a representative device
according to some embodiments of the present invention.
DESCRIPTION OF EMBODIMENTS
[0064] Before compositions and methods provided herein are
described, it is to be understood that the embodiments are not
limited to the particular processes, compositions, or methodologies
described, as these may vary. It is also to be understood that the
terminology used in the description is for the purpose of
describing some embodiments, and is not intended to limit the scope
of the embodiments.
[0065] Various methods and embodiments are described herein. The
methods and embodiments can be combined with one another. The
definitions and embodiments described herein are not limited to a
particular method or example unless the context clearly indicates
that it should be so limited.
[0066] As used herein, the phrase "detection of an analyte,"
"detecting an analyte" refers the detection of multiple analytes
with a single signal. The detection of multiple analytes can be, as
described herein, at least, or exactly, 2, 3, 4, or 5 analytes with
a single signal.
[0067] It must be noted that, as used herein and in the appended
claims, the singular forms "a", "an" and "the" include plural
reference unless the context clearly dictates otherwise. Unless
defined otherwise, all technical and scientific terms used herein
have the same meanings as commonly understood by one of ordinary
skill in the art. Although any methods similar or equivalent to
those described herein can be used in the practice or testing of
embodiments of the present invention, the preferred methods are now
described. All publications mentioned herein are incorporated by
reference in their entirety to the extent to support the presently
described subject matter. Nothing herein is to be construed as an
admission that the subject matter is not entitled to antedate such
disclosure by virtue of prior invention.
[0068] As used herein, the term "about" means plus or minus 10% of
the numerical value of the number with which it is being used.
Therefore, about 50% means in the range of 45%-55%. Additionally,
in phrase "about X to Y," is the same as "about X to about Y," that
is the term "about" modifies both "X" and "Y."
[0069] As used herein, the term "optional" or "optionally" means
that the subsequently described structure, event or circumstance
may or may not occur, and that the description includes instances
where the event occurs and instances where it does not.
[0070] As used herein, the term "sample" means any fluid medium or
liquid that may contains a particular item (e.g. analyte) or
suspected of containing a particular item. In some embodiments,
samples may be used which are high in dissolved solids without
further processing, and samples containing high solids
(non-dissolved) may be analyzed, in some embodiments, through the
use of a filter or used in conjunction with additional manual
steps. Samples may also be non-filtered or purified prior to being
used in a method or device described herein. Samples may be a
liquid, a suspension, extracted or dissolved sample, or a
supercritical fluid. If a sample is going to be used in a flow
device (vertical or lateral) some flow properties must exist in the
sample or extract to allow flow through the devices and systems
described herein. Examples of samples include, but are not limited
to, blood, food swabs, food extracts, food suspensions, food
cultures, bacterial cultures, viral cultures, amplification
reactions, saliva, biological fluid, PCR reactions, and the like.
The sample can also be derived from a another sample. For example,
a PCR reaction can be performed on a nucleic acid mixture that has
been extracted, isolated, and/or purified from another sample
(e.g., food, cellular, viral, bacterial, blood, and the like). The
PCR reaction would be considered to be a sample derived from
another sample.
[0071] A "food suspension" refers to raw or cooked food that has
been placed or suspended in a solution. The food solution may be
mixed, vortexed or blended. A "food culture" is a food sample that
is cultured under conditions to enrich the sample. This process can
also be referred to as "enrichment." The enrichment can be used to
facilitate sample analysis to better detect the presence or absence
of multiple analytes with a single signal. The sample can also be a
reaction sample that is derived from a different sample. An example
of a reaction sample is an "enrichment." For example, a blood or
food sample may be processed (e.g. cultured, purified, separated
into components, and the like) and the processed sample can be
tested for the detection of multiple analytes. In some embodiments,
two analytes are detected in a blood sample or a food sample. In
some embodiments, the analytes can be detected by performing two
amplification reactions that are specific for the two analytes and
then the two amplification products can be detected with a single
signal to detect the presence of the two analytes in a sample
concurrently. In some embodiments, three analytes are detected
using a single signal. The detection can be concurrent, that is the
signal is only generated when all the analytes are present in the
same sample. The concurrent signal generation can be effectuated
through the creation of a bridging complex, which is described
herein. Non-limiting embodiments of the bridging complex can be
seen in FIGS. 1-3.
[0072] As used herein, the term "solid support" means a material
that is substantially insoluble in a selected system, or which can
be readily separated (e.g., by precipitation) from a selected
system in which it is present. Solid supports useful in practicing
the present methods can include groups that are activated or
capable of activation to allow certain compounds or molecules (e.g.
capture reagents, antibodies, and the like) to be bound to the
solid support. The solid support may, for example, be agarose,
sepharose, polyacrylamide, agarose/polyacrylamide co-polymers,
dextran, cellulose, polypropylene, polycarbonate, nitrocellulose,
glass paper, or any other suitable substance capable of providing a
suitable solid support. In some embodiments, the solid support may
be in the form of granules, a powder or a gel suitable for use in
chromatography. The solid support can also be a membrane, such a
nitrocellulose, PVC, and the like. Other types of membranes can
also be used and there is no specific requirement for the type of
membrane that can be used. In some embodiments, the solid support
is a test membrane. Examples of test membranes are described
herein.
[0073] As used herein, the term "analyte" includes, but is not
limited to, antigens, nucleic acid molecules encoded by a cell,
virus, bacteria or other type of microorganism, amplification
products (e.g. amplicons), a peptide, a sugar, and the like. In
some embodiments, the analyte is not an antibody or functional
fragment thereof. Nucleic acid molecules can be detected as
described herein by using the methods described herein in
combination with other known methods or devices, such as
amplification methods (e.g. PCR, RT-PCR, and the like),
hybridization methods, labeled primers, and the like. The term
"target molecule" can be used interchangeably with the term
"analyte." The amplification methods can be used to amplify the
amount of nucleic acid molecules present in a sample to facilitate
the detection of the analyte. Other types of analytes that can be
detected using the methods described herein include, but are not
limited to antigens, antibodies, receptors, ligands, chelates,
proteins, enzymes, nucleic acids, DNA, RNA, pesticides, herbicides,
inorganic or organic compounds, or any material for which a
specific binding reagent may be found. The analyte can also refer
to different epitopes present on the same protein or polypeptide.
The analyte can also refer to analytes from pathogenic or
non-pathogenic organisms. The analytes can also be referred to as
an analyte of interest in a sample. That is, the analyte can be
referred to as an agent that a user is determining the presence or
absence of in a sample.
[0074] As discussed herein, the analyte can be an amplification
product, such as a product of a PCR reaction. The PCR product is
amplifying a nucleic acid sequence from a test sample. Thus,
detection of the PCR product in sample is determining whether the
nucleic acid sequence that the PCR product is based upon is present
in the initial sample. For example, if one of skill in the art is
determining whether a food sample is contaminated with E. Coli,
nucleic acid sequences that are specific for E. Coli can be
amplified (e.g. by PCR) and then detected according to the methods
described herein. The detection of the amplification products (i.e.
amplicons) indicates that the food sample contained the native
nucleic acid sequences that are specific for E. Coli. This is
example is non-limiting and can be applied to detecting other
nucleic acid sequence or other types of analytes present in a
native sample. The analyte can be what is in the initial sample or
an analyte that is derived from the initial sample by, for example,
using PCR. When the plurality of analytes is being detected with a
single signal according to the methods provided herein, the
analytes can also have heterologous tags or interaction units, and
the modified analyte is also referred to as the analyte. In some
embodiments, the analyte will be free of heterologous interaction
units, such as fluorescent tags, biotin, digoxigenin, and the
like.
[0075] An analyte is different from a reagent that is used to
detect the presence or absence of an analyte. Thus, a reagent that
is added to the sample to determine if the analyte is present is
not an analyte of interest. For example, in a typical sandwich
assay, a first antibody is attached to a solid support. The solid
support coated with an antibody is contacted with a sample to
determine the presence or absence of an antigen that binds to the
antibody. A secondary antibody is then also added to detect the
antigen. The presence of the secondary antibody is then often
detected by the addition of a third antibody that has, for example.
an enzyme conjugated to it so that it can be detected through
various means (e.g. HRP-linked antibodies). The secondary antibody
is not an analyte of interest because it is a reagent used to
detect the primary antigen. Therefore, a sandwich assay does not
detect the presence of a plurality of analytes with a single signal
according to the methods described herein because the secondary
antibody is a reagent, or tool, to detect the presence or absence
of the antigen, or an analyte of interest. An analyte is also not a
component or portion that is found on a bridging entity. For
example, in U.S. Published Application No. 2010/0273145 FIGS. 1 and
2 show an analyte binding to a bridging entity, which then binds to
a signaling entity to detect the presence of the analyte. Neither
the bridging unit, or any portion thereof, or the signaling entity
is an analyte or analyte of interest. These components are reagents
used to detect the analyte, which in the case of U.S. Published
Application No. 2010/0273145 is the detection of a single analyte.
U.S. Published Application No. 2010/0273145 is hereby incorporated
by reference with regards to its figures and the explanation of
their components.
[0076] In some embodiments, the analyte is a protein, such as a
pathogen protein. A pathogen protein refers to a protein that is
from a pathogen. Examples of pathogens include, but are not limited
to, viruses, prokaryotes and, for example, pathogenic eukaryotic
organisms such as unicellular pathogenic organisms and
multicellular parasites. Pathogens also can include protozoan
pathogens which include a stage in the life cycle where they are
intracellular pathogens. As used herein, the term "intracellular
pathogen" means a virus or pathogenic organism that, at least part
of its reproductive or life cycle, exists within a host cell and
therein produces or causes to be produced, pathogen proteins. A
pathogen can also be a food-borne pathogen.
[0077] Bacterial pathogens include, but are not limited to, such as
bacterial pathogenic gram-positive cocci, which include but are not
limited to: pneumococcal, staphylococcal, and streptococcal.
Pathogenic gram-negative cocci include, but are not limited to:
meningococcal and gonococcal. Pathogenic enteric gram-negative
bacilli include, but are not limited to: enterobacteriaceae,
pseudomonas, acinetobacteria, eikenella, melioidosis, salmonella,
shigellosis, hemophilus, chancroid, brucellosis, tularemia,
yersinia (pasteurella), streptobacillus moniliformis, spirilum,
Listeria monocytogenes, Erysipelothrix rhusiopathiae, diphtheria,
cholera, anthrax, donovanosis (granuloma inguinale), and
bartonellosis. Pathogenic anaerobic bacteria include, but are not
limited to, those that are responsible for: tetanus, botulism,
other clostridia, tuberculosis, leprosy, and other mycobacteria.
Pathogenic spirochetal diseases include, but are not limited to:
syphilis, treponematoses, yaws, pinta and endemic syphilis, and
leptospirosis. Other infections caused by higher pathogen bacteria
and pathogenic fungi include, but are not limited to:
actinomycosis, nocardiosis, cryptococcosis, blastomycosis,
histoplasmosis, and coccidioidomycosis, candidiasis, aspergillosis,
mucormycosis, sporotrichosis, paracoccidiodomycosis,
petriellidiosis, torulopsosis, mycetoma, chromomycosis, and
dermatophytosis. Rickettsial infections include, but are not
limited to, rickettsia and rickettsioses. Examples of mycoplasma
and chlamydial infections include, but are not limited to:
mycoplasma pneumonia, lymphogranuloma venereum, psittacosis, and
perinatal chlamydial infections. Pathogenic protozoans and
helminths and infectious eukaryotes thereby include, but are not
limited to: amebiasis, malaria, leishmaniasis, trypanosomiasis,
toxoplasmosis, pneumocystis carinii, babesiosis giardiasis
trichinosis filariasis schistosomiasis, nematodes, trematodes or
flukes, and cestode (tapeworm) infections. Bacteria also include,
but are not limited to, Listeria, E. coli, Campylobacter species,
and Salmonella species. In some embodiments, E. coli is E. coli
0157.
[0078] Examples of viruses include, but are not limited to, HIV,
Hepatitis A, B, and C, FIV, lentiviruses, pestiviruses, West Nile
Virus, measles, smallpox, cowpox, ebola, coronavirus, and the like.
Other pathogens are also disclosed in U.S. Patent Application
Publication No. 20080139494, which are incorporated herein by
reference.
[0079] In some embodiments, the pathogen is a food borne pathogen.
The analyte can be present on a food borne pathogen. Food borne
pathogens are pathogens (e.g. viral or bacterial) that cause
illness after eating contaminated food. The food itself does not
directly cause the illness, but it is rather the consumption of the
food borne pathogen that is present on the food that causes the
illness. In some embodiments, the food borne pathogen is E. coli,
Listeria, a Campylobacter species, or a Salmonella species. In some
embodiments, the analyte is chosen from a food borne pathogen
analyte. For example, the food borne pathogen analyte can be, but
is not limited to, chosen from an E. coli analyte, a Listeria
analyte, a Campylobacter species analyte, or a Salmonella species
analyte. In some embodiments, the analyte is the specific
O-Antigen. In some embodiments, the O-antigen is the E. coli
antigen and/or a Salmonella species O-antigen and can be used for
E. coli and Salmonella detection. In some embodiments, the analyte
is a flagellin antigen. In some embodiments, the analyte is the
Campylobacter flagellin antigen. In some embodiments the analyte is
a virulence factor gene such as the Shiga toxin gene amplified from
pathogenic E. coli or Salmonella. In some embodiments, the analyte
is a DNA or RNA sequence that is amplified via an amplification
method (e.g. PCR or RT-PCR) and then detected according to the
methods described herein.
[0080] As described herein, an analyte can be an amplification
product. The amplification product, such as PCR product (e.g. a
double stranded PCR product), can be labeled with interaction
units. The production of a labeled amplification product with the
units can be made by the use of primers labeled or conjugated with
the two interaction units. In some embodiments, an analyte will
have two different interaction units so that the bridging complex
can be assembled and the detection of multiple analytes is possible
through a signal detection unit.
[0081] As used herein, the term "signal detection unit" means a
unit that can be detected to determine if the analyte or analytes
are present in a sample. The signal detection unit can be any
reagent or composition that can be detected. In some embodiments,
the signal detection unit is attached to a capture reagent. Thus,
the signal detection unit can be used to detect the presence of the
capture reagent binding to its specific binding partner. The
capture reagent can comprise a detection reagent directly or the
capture reagent can further comprise a particle that comprises the
detection reagent. In some embodiments, the capture reagent and/or
particle comprises a color, colloidal gold, a radioactive tag, a
fluorescent tag, or a chemiluminescent substrate. In some
embodiments, the signal detection unit comprises a near-infrared or
infrared tag or substrate. In some embodiments, the signal
detection unit comprises a color, colloidal gold, a radioactive
tag, a fluorescent tag, or a chemiluminescent substrate. In some
embodiments, the signal detection unit comprises a nanocrystal,
functionalized nanoparticles, up-converting nanoparticles, cadmium
selenide/cadmium sulfide fusion nanoparticles, quantum dots, and a
Near-Infrared (NIR) fluorophore or material (such as, but not
limited to, materials such as lanthanide clusters and
phthalocyanines, as well as light emitting-diodes consisting of
CuPc, PdPc, and PtPc) capable of emitting light in the NIR
spectrum. In some embodiments, a capture reagent and/or particle is
conjugated to the signal detection unit, such as but not limited
to, colloidal gold, silver, radioactive tag, fluorescent tag, or a
chemiluminescent substrate, near-infrared compound (e.g. substrate,
molecule, particle), or infrared compound (e.g. substrate,
molecule, particle), nanoparticle, emissive nanoparticle, quantum
dot, magnetic particle, or an enzyme.
[0082] The signal detection unit can also be, for example, a viral
particle, a latex particle, a lipid particle, a fluorescent
particle, a near-infrared particle, or infrared particle. As used
herein, the term "fluorescent particle" means a particle that emits
light in the fluorescent spectrum. As used herein, the term
"near-infrared particle" means a particle that emits light in the
near-infrared spectrum. As used herein, the term "infrared
particle" means a particle that emits light in the infrared
spectrum. In some embodiments, the colloidal gold has a diameter
size of: about 20 nm, about 30 nm, or about 40 nm, or in the range
of about 20-30 nm, about 20-40 nm, about 30-40 nm, or about 35-40
nm. In some embodiments, the particle comprises a metal alloy
particle. In some embodiments, the metal alloy particle has a
diameter from about 10 to about 200 nm. Examples of metal alloy
particles include, but are not limited to, gold metal alloy
particles, gold-silver bimetallic particles, silver metal alloy
particles, copper alloy particles, Cadmium-Selenium particles,
palladium alloy particles, platinum alloy particles, and lead
nanoparticles.
[0083] As discussed herein the signal detection can will bind to
one of the analytes. A non-limiting example of the signal detection
unit binding to an analyte is shown in FIG. 1. FIG. 1, which is
described in more detail herein, shows the signal detection unit 60
binding to the analyte 40 through a capture reagent 50. However,
the signal detection unit can also bind to other portions of the
complex. Any component that is necessarily present only when both
the plurality of analytes is present in the complex can be a
binding partner for the signal detection unit. Often, but not
exclusively this will be one of the analytes, but can also be a
capture reagent that is bound to the analyte. In contrast, in some
embodiments, the signal detection unit does not bind solely to the
analyte that is bound to the solid support, the solid support, or
the capture reagent bound directly to the solid support, if present
on the solid support. For example, in FIG. 1, the signal detection
unit will not bind directly to the solid support 10, the capture
reagent 15, or the analyte 20. Without being bound to any
particular theory, if the signal detection unit binds directly with
the solid support 10, the capture reagent 15, or the analyte 20,
the method would provide a false positive as the signal would be
detected without the plurality of analytes necessarily being
present. For example, FIG. 6, illustrates the signal detection unit
binding to a component of a multi-component bridging unit.
Embodiments of the bridging unit, and a multi-component bridging
unit, are described herein and, for example, with references to
FIGS. 4 and 5. FIG. 6 illustrates a signal detection unit 60 with
its capture reagent 50 binding to a component of the bridging unit
30. The bridging unit comprises 30 a particle 34, a first capture
reagent 31, a second capture reagent 32, and a third capture
reagent 33. FIG. 6 illustrates the signal detection unit binding to
the second capture reagent 32. The capture reagent 32 will only be
present in the complex if both analytes are present in the complex.
If capture reagent 32 is not present this means that there is no
bridged complex of the plurality of analytes. Therefore, the signal
detection unit will only be part of the complex if the plurality of
analytes are present in the complex, thus avoiding false positives.
If both analytes are not present the capture reagent 32 will not be
part of the complex, and, therefore, there will be no binding
partner for the signal detection unit. Accordingly, the signal
detection unit will only be detectable when the plurality of
analytes are present. Therefore, in some embodiments, the signal
detection unit binds to any component that is only present when the
plurality of analytes are also present. Other properties,
characteristics, and structural features of the multi-component
bridge unit are also disclosed herein and are readily apparent
based upon the present disclosure.
[0084] Examples of devices in which the presently described methods
can be used are described in, for example, in U.S. Pat. No.
8,012,770, U.S. patent application Ser. No. 13/360,528, filed Jan.
27, 2012, PCT Publication No. WO 2011/044574, each of which is
incorporated herein by reference in its entirety. The presently
describes methods, however, can be used with any number of devices
or formats, such as multi-well plates, arrays, microarrays, or in
an "ELISA" type format. Examples of devices are also described
herein, but these examples are non-limiting. The methods described
herein can also be used in conjunction with lateral flow devices.
In a lateral flow device the different portions of the device are
in the same plane as opposed to a vertical flow device.
Non-limiting examples of the lateral flow devices can be found in
U.S. Pat. Nos. 6,485,982, 6,818,455, 6,951,631, 7,109,042,
RE39,664, and the like, each of which are hereby incorporated by
reference. The lateral flow devices can be adapted for the methods
described herein as they are described for the vertical flow
devices. In a lateral flow device, the region that indicates a
positive or negative result can comprise the capture reagent that
binds to one of the analytes. The bridge unit can either be present
in one of the lateral flow regions or mixed with the analytes
before addition to the device--this can also be done for other
devices and solid supports. The signal detection unit can also be
incorporated into one of the lateral flow regions. As is clear from
the present disclosure the type of device or solid support is not
critical and the methods can be adapted based upon the examples and
embodiments described herein.
[0085] As used herein, the term "amplicon" means an amplification
product such as a nucleic acid molecule that is amplified by a PCR
reaction or other amplification reaction or method. As discussed
herein, an amplicon can be an analyte. The amplicon can be a
double-stranded nucleic acid molecule. The amplification product
can be detected directly or indirectly through the use of
antibodies or other capture reagent systems, including those that
are described herein. The amplification product can also be
detected through hybridization methods in whole or in part as
described herein. The amplification product can also be produced,
for example, through RT-PCR or linear amplification.
[0086] In some embodiments, the amplicon is a PCR product. The PCR
reaction products (e.g. amplicons) can be labeled such that they
are detectable either by another antibody or antibody like system,
such as but not limited to biotin-avidin/streptavidin system,
systems, hapten systems, BRDU labeling of DNA, intercalating agents
that label DNA, labeled dNTPS, and the like can also be used where
the PCR products are labeled. The analyte, which can, for example,
but not limited to, be a nucleic acid (single stranded or double
stranded) and can be recognized or detected with an antibody or
other capture reagent system, such as those described herein. The
nucleic acid molecule can be labeled with a biotin label or other
type of label that can be detected using a method described herein.
Other examples of labels include fluorescent labels. The
fluorescent labels can be for example, fluorescein (e.g.
fluorescein isothiocyanate (FITC)), rhodamine (e.g.
tetramethylrhodamine (TAMRA)), and the like. The amplicons can be
generated with these labels by using labeled primers. The labels
can be incorporated into the amplicon through the amplification
procedure and, thus, become part of the analyte. The labels would
be considered heterologous tags because the labels are not found in
the native sequence that is used as the template for the amplicon.
Capture reagents (e.g. antibodies) can be used that bind to the
labels to help in forming the complexes that are described herein,
which enable the detection of multiple analytes with a single
signal. These labels can act as interaction units. A non-limiting
example of how the labels can act as interaction units such that
multiple analytes can be detected with a single signal is shown in
FIG. 3.
[0087] For example, in some embodiments, a PCR reaction is
performed with a hapten and/or biotin labeled DNA or RNA primers
with homology to an analyte nucleic acid sequence. The analyte
nucleic acid sequence can be, but not limited to, a toxin gene
and/or a toxin molecule (e.g. Shiga toxin) from a meat sample. The
sample, however, can be any sample, and the analyte can be any
other type of analyte described herein. The PCR reactions can be
performed to produce multiple analytes with the interaction units.
Following amplification with the primers, the PCR sample can be
detected using a method described herein. The PCR reaction can also
be performed with digoxigenin and/or TAMRA and/or with FITC and
TAMRA labeled primers. These can create the differentially labeled
amplicons that can be bridged together through the use of capture
reagents to enable the detection of multiple analytes with a single
signal. An example of such a complex is shown in FIG. 3.
[0088] FIG. 3 illustrates a test membrane (i.e., solid support 10)
with an Anti-Dig antibody (i.e., capture reagent 15), a
Digoxigenin/TAMRA labeled amplicon (i.e., a first analyte 20, a
first interaction unit 21, and a second interaction unit 22), an
anti-rhodamine antibody ((i.e. bridge unit 30), a FITC/TAMRA
labeled amplicon (i.e., a second analyte 40, a first interaction
unit 41, and a second interaction unit 42); and a streptavidin-gold
complex binding to a biotinylated anti-FITC antibody (i.e., a
signal generation unit 60 and a third capture reagent 50).
[0089] Briefly, after the PCR reactions are performed, the
amplicons can be contacted with a solid support, a bridging unit,
and a signal detection unit. The solid support can have a capture
reagent that binds to an interaction unit on the first analyte. The
bridging unit can have, or be, a capture reagent that binds to
interaction units on the first and second analytes such that the
binding to the interaction units on the first and second analytes
brings the analytes together into a complex. The signal detection
unit can bind to an interaction unit present on the one of the
second analyte. The signal detection unit can then emit a
detectable signal or the signal detection unit can be detected by
the addition of another detection system. For example, in FIG. 3,
the signal detection unit is a capture reagent (e.g. antibody) that
binds to the interaction unit on the second analyte. The signal
detection unit is biotinylated. The presence of the signal
detection unit can be then be determined by the addition of
streptavidin. The streptavidin will only bind to a complex that has
both analytes present. In the non-limiting example shown in FIG. 3,
the streptavidin is labeled with colloidal gold which enables the
detection. However, other labels or detection systems could be used
to detect the streptavidin. In the embodiments of the vertical flow
devices described herein, the test membrane is the solid support
with the capture reagent, and the conjugate pad can comprise the
signal detection unit or the molecule that detects the binding of
the signal detection unit to the interaction unit of the second
analyte.
[0090] FIG. 7 illustrates a non-limiting work flow procedure that
could be used to detect a plurality of analytes with a single
signal using amplicons to detect the presence of an analyte of
interest in a sample. A food sample 7000 is analyzed to determine
the presence or absence of pathogenic E. Coli. The food sample 7000
is processed (e.g. enriched, cultured, nucleic acid, purification,
isolation, extraction, or other similar steps) to extract, isolate
or otherwise make available the nucleic acids present in the food
sample. The nucleic acid sequences present in the processed sample
7001 can be amplified, such as but not limited to by PCR, to
amplify the specific pathogenic E. Coli sequences. Examples of
these sequences are described herein. No specific primer set need
be used as those can be modified based upon the target sequence to
be amplified. As described herein, the primers can be labeled,
thereby creating labeled amplicons (analytes with heterologous
interaction units). The first analyte 7020 and the second analyte
7040 will be generated if the target sequences are present in the
food sample and the processed sample. The analytes are shown with
heterologous interaction units (7021, 7022, 7041, and 7042). The
analytes can be mixed with a bridge unit 7030. The mixture will
form a bridged complex 7100. The analytes can then be detected by
contacting the bridged complex with a solid support 7010 comprising
a capture reagent 7015 and a signal detection unit 7060 comprising
a capture reagent 7050. As discussed herein, the a signal detection
unit 7060 comprising a capture reagent 7050 can be absorbed onto a
membrane and allowed to interact with the bridged complex. The
solid support 7010 comprising a capture reagent 7015 can be a test
membrane with an antibody. These elements can be incorporated into
a device as described herein. Although FIG. 7 shows the steps being
performed separately they can also be performed in different order
and some steps may be combined. For example, the step of mixing the
analytes with the bridge unit can also be combined with contacting
the analytes with the signal detection unit comprising a capture
reagent. The detection step of adding to the complex to the solid
support could then be done subsequently. In some embodiments, the
analytes, bridge unit, signal interaction unit comprising a capture
reagent, and the solid support comprising a capture reagent can be
mixed together simultaneously or nearly simultaneously and then the
signal detection unit can be detected. The signal detection unit
will only be detected or detected above background levels (i.e.
above a negative control) when the plurality of the analytes are
present in the sample being tested. That is, in FIG. 7, the complex
7200 will only be formed if both analytes, and thus both target
sequences are present in the food sample 7000, are present. The
complex 7200 will not be formed if one of the analytes is missing.
The workflow shown in FIG. 7 can also include a washing step to
wash away any unbound material or components that do not form a
complex 7200. Washing steps may also be incorporated into any
method described herein.
[0091] In some embodiments of the methods described herein, the
method of detecting a plurality of analytes with a single signal
comprises amplifying a plurality of target nucleic acid sequences
present in a sample. The target sequences can be the analytes or
the amplified products can be the analytes. The detection of the
amplified sequences (e.g., PCR products) indicates the presence of
the template sequences in the original sample.
[0092] In some embodiments, methods of concurrently detecting a
plurality of analytes with a single signal comprise a) contacting a
device for detecting a plurality of analytes with a single signal
with one or more samples comprising a plurality of analytes; and
detecting the presence or absence of the signal detection unit
which indicates the presence or absence of the first analyte of
interest and second analyte of interest concurrently. The device
can be any device used to detect the presence or absence of analyte
including, but not limited to the devices described herein. In some
embodiments, the device comprises: a housing comprising: an inlet
opening in fluid contact with a conjugate pad; a force member; a
slidable locking member contacting the force member; an attachment
member contacting the force member; a sliding button contacting the
attachment member; and a detection membrane system comprising the
conjugate pad, a test membrane, and an absorbent member, at least a
portion of the conjugate pad, test membrane, and absorbent member
are substantially parallel to each other, the force member contacts
the detection membrane system and is capable of applying pressure
substantially perpendicular to the detection membrane system, the
sliding button moves the slidable locking member, the conjugate pad
comprises a signal detection unit comprising a third capture
reagent; the test membrane comprises a first capture reagent
affixed to the test membrane.
[0093] In some embodiments, the one or more samples comprises a
first analyte of interest, a second analyte of interest, and a
bridge unit comprising a second capture reagent, wherein the first
analyte of interest comprises a first interaction unit that binds
to the first capture reagent and a second interaction unit that
binds to the bridge unit, and the second analyte of interest
comprises a first interaction unit that binds the bridge unit and a
second interaction unit. In some embodiments, the signal detection
unit comprises the third capture reagent that binds to the second
analyte, to the second analyte's first interaction unit or second
interaction unit, to a component of the first and second analyte
complex, or to a component of the bridge unit that that is only
present when the complex contains the first and second
analytes.
[0094] In some embodiments, the detecting comprises moving the
conjugate pad after a portion of the one or more samples has
contacted and flowed through the conjugate pad, thereby exposing at
least a portion of the test membrane for detection of the signal
detection unit to indicate the presence or absence of the plurality
of analytes with a single signal. In some embodiments, the
conjugate pad is moved by moving the slidable locking member. In
some embodiments, the one or more samples are contacted with the
conjugate pad prior to compressing the detection membrane system.
The method can be performed with multiple samples to detect the
plurality of analytes. For example, if a plurality of amplification
reactions are performed to produce a plurality of amplicons
(analytes) each of the plurality of amplifications reactions is
considered a separate sample. To detect the plurality of analytes
with a single signal the samples have to be mixed. The plurality of
samples can be mixed prior to contacting the device or be contacted
with the device (solid support) sequentially, or
simultaneously.
[0095] In some embodiments, the first and second analyte are
amplicons. In some embodiments, the first and second analytes are
PCR reaction products. In some embodiments, the first analyte's
first interaction unit is a digoxigenin label. In some embodiments,
the first analyte's second interaction unit is a rhodamine label.
In some embodiments, the second analyte's first interaction unit is
a rhodamine label. In some embodiments, the second analyte's second
interaction unit is a fluorescein label. In some embodiments, the
third capture reagent binds to the second analyte's second
interaction unit. In some embodiments, the third capture reagent is
a biotinylated capture reagent. In some embodiments, the signal
interaction unit is coated with streptavidin. In some embodiments,
the signal interaction unit is streptavidin coated colloidal gold.
In some embodiments, the first and second analytes are nucleic acid
amplification products, wherein: the first analyte comprises a
digoxigenin label and a rhodamine label; the second analyte
comprises a rhodamine label and a fluorescein label; the first
capture reagent is an anti-digoxigenin label antibody; the second
capture reagent is an anti-rhodamine label antibody; the third
capture reagent is a biotinylated anti-fluorescein label antibody;
and the signal interaction unit is streptavidin coated colloidal
gold.
[0096] As used herein and throughout, the terms "attached" or
"attachment" can include both direct attachment or indirect
attachment. Two components that are directly attached to one
another are also in physical contact with each other. Two
components that are indirectly attached to one another are attached
through an intermediate component. For example, Component A can be
indirectly attached to Component B if Component A is directly
attached to Component C and Component C is directly attached to
Component B. Therefore, in such an example, Component A would be
said to be indirectly attached to Component B.
[0097] The term "capture reagent" means a reagent capable of
binding a target molecule or analyte to be detected in a sample.
Examples of capture reagents include, but are not limited to,
antibodies or antigen binding fragments thereof, an
oligonucleotide, and a peptoid. Other examples of capture reagents
include, but are not limited to, small molecules or proteins, such
as biotin, avidin, streptavidin, hapten, digoxigenin, BRDU, single
and double strand nucleic acid binding proteins or other
intercalating agents, and the like, or molecules that recognize and
capture the same. These are non-limiting examples of capture
reagents. Other types of capture reagents can also be used.
[0098] As discussed herein, a capture reagent can also refer to,
for example, antibodies. Intact antibodies, also known as
immunoglobulins, are typically tetrameric glycosylated proteins
composed of two light (L) chains of approximately 25 kDa each, and
two heavy (H) chains of approximately 50 kDa each. Two types of
light chain, termed lambda and kappa, exist in antibodies.
Depending on the amino acid sequence of the constant domain of
heavy chains, immunoglobulins are assigned to five major classes:
A, D, E, G, and M, and several of these may be further divided into
subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and
IgA2. Each light chain is composed of an N-terminal variable (V)
domain (VL) and a constant (C) domain (CL). Each heavy chain is
composed of an N-terminal V domain (VH), three or four C domains
(CHs), and a hinge region. The CH domain most proximal to VH is
designated CH1. The VH and VL domains consist of four regions of
relatively conserved sequences named framework regions (FR1, FR2,
FR3, and FR4), which form a scaffold for three regions of
hypervariable sequences (complementarity determining regions,
CDRs). The CDRs contain most of the residues responsible for
specific interactions of the antibody or antigen binding protein
with the antigen. CDRs are referred to as CDR1, CDR2, and CDR3.
Accordingly, CDR constituents on the heavy chain are referred to as
H1, H2, and H3, while CDR constituents on the light chain are
referred to as L1, L2, and L3. CDR3 is the greatest source of
molecular diversity within the antibody or antigen binding
protein-binding site. H3, for example, can be as short as two amino
acid residues or greater than 26 amino acids. The subunit
structures and three-dimensional configurations of different
classes of immunoglobulins are well known in the art. For a review
of the antibody structure, see Antibodies: A Laboratory Manual,
Cold Spring Harbor Laboratory, Eds. Harlow et al., 1988. One of
skill in the art will recognize that each subunit structure, e.g.,
a CH, VH, CL, VL, CDR, and/or FR structure, comprises active
fragments. For example, active fragments may consist of the portion
of the VH, VL, or CDR subunit that binds the antigen, i.e., the
antigen-binding fragment, or the portion of the CH subunit that
binds to and/or activates an Fc receptor and/or complement.
[0099] Non-limiting examples of binding fragments encompassed
within the term "antigen-specific antibody" used herein include:
(i) an Fab fragment, a monovalent fragment consisting of the VL,
VH, CL and CH1 domains; (ii) an F(ab')2 fragment, a bivalent
fragment comprising two Fab fragments linked by a disulfide bridge
at the hinge region; (iii) an Fd fragment consisting of the VH and
CH1 domains; (iv) an Fv fragment consisting of the VL and VH
domains of a single arm of an antibody, (v) a dAb fragment, which
consists of a VH domain; and (vi) an isolated CDR. Furthermore,
although the two domains of the Fv fragment, VL and VH, are coded
for by separate genes, they may be recombinantly joined by a
synthetic linker, creating a single protein chain in which the VL
and VH domains pair to form monovalent molecules (known as single
chain Fv (scFv)). The most commonly used linker is a 15-residue
(Gly.sub.4Ser).sub.3 peptide, but other linkers are also known in
the art. Single chain antibodies are also intended to be
encompassed within the terms "antibody or antigen binding protein,"
or "antigen-binding fragment" of an antibody. The antibody can also
be a polyclonal antibody, monoclonal antibody, chimeric antibody,
antigen-binding fragment, Fc fragment, single chain antibodies, or
any derivatives thereof. The capture reagent or antibody can also
be a VHH region, a bi-specific antibody, a peptide fragment
comprising an antigen binding site, or a compound that binds to an
antigen of interest. The antigen of interest can be an amplicon or
other type of analyte.
[0100] These antibodies can be purchased or obtained using
conventional techniques known to those skilled in the art, and the
fragments are screened for utility in the same manner as intact
antibodies. Antibody diversity is created by multiple germline
genes encoding variable domains and a variety of somatic events.
The somatic events include recombination of variable gene segments
with diversity (D) and joining (J) gene segments to make a complete
VH domain, and the recombination of variable and joining gene
segments to make a complete VL domain. The recombination process
itself is imprecise, resulting in the loss or addition of amino
acids at the V(D)J junctions. These mechanisms of diversity occur
in the developing B cell prior to antigen exposure. After antigenic
stimulation, the expressed antibody genes in B cells undergo
somatic mutation. Based on the estimated number of germline gene
segments, the random recombination of these segments, and random
VH-VL pairing, up to 1.6.times.10.sup.7 different antibodies may be
produced (Fundamental Immunology, 3rd ed. (1993), ed. Paul, Raven
Press, New York, N.Y.). When other processes that contribute to
antibody diversity (such as somatic mutation) are taken into
account, it is thought that upwards of 1.times.10.sup.10 different
antibodies may be generated (Immunoglobulin Genes, 2nd ed. (1995),
eds. Jonio et al., Academic Press, San Diego, Calif.). Because of
the many processes involved in generating antibody diversity, it is
unlikely that independently derived monoclonal antibodies with the
same antigen specificity will have identical amino acid
sequences.
[0101] Antibody or antigen binding protein molecules capable of
specifically interacting with the antigens, epitopes, or other
molecules described herein may be produced by methods well known to
those skilled in the art. For example, monoclonal antibodies can be
produced by generation of hybridomas in accordance with known
methods. Hybridomas formed in this manner can then be screened
using standard methods, such as enzyme-linked immunosorbent assay
(ELISA) and Biacore analysis, to identify one or more hybridomas
that produce an antibody that specifically interacts with a
molecule or compound of interest.
[0102] As an alternative to preparing monoclonal antibody-secreting
hybridomas, a monoclonal antibody to a polypeptide of the present
invention may be identified and isolated by screening a recombinant
combinatorial immunoglobulin library (e.g., an antibody phage
display library) with a polypeptide of the present invention to
thereby isolate immunoglobulin library members that bind to the
polypeptide. Techniques and commercially available kits for
generating and screening phage display libraries are well known to
those skilled in the art. Additionally, examples of methods and
reagents particularly amenable for use in generating and screening
antibody or antigen binding protein display libraries can be found
in the literature.
[0103] The term "capture reagent" also includes chimeric
antibodies, such as humanized antibodies, as well as fully
humanized antibodies. In some embodiments the capture reagent is a
Goat anti-E. coli 0157:H7 antibody Cat #: 70-XG13 (Fitzgerald
Industries); E. coli 0157:H7 mono Cat #: 10-E13A(Fitzgerald
Industries); E. coli 0157:H7 Cat #: 10C-CR1295M3(Fitzgerald
Industries); E. coli 0157:H7 mono Cat #: 10-E12A(Fitzgerald
Industries); or Goat anti-mouse IgG Cat #: ABSE-020 (DCN). The
capture reagent can also be, for example, protein A, protein G, and
the like. The capture reagent can also be an antibody that binds or
specifically binds to a fluorescent label (e.g. fluorescein or
rhodamine), a hapten, digoxigenin and the like. A capture reagent,
such a streptavidin can be conjugated with colloidal gold. The
streptavidin-gold complex can then be used, for example, to bind to
a biotinylated product, such as a biotinylated antibody. A
non-limiting example can be seen in FIG. 3. The labels shown in
FIG. 3 are for illustrative purposes only and other permutations
can be used.
[0104] The capture reagent can also include an anti-antibody, i.e.
an antibody that recognizes another antibody but is not specific to
an analyte, such as, but not limited to, anti-IgG, anti-IgM, or
ant-IgE antibody.
[0105] As used herein, the term "concurrently" refers to the
detection of multiple analytes simultaneously or nearly
simultaneously. As used herein, "A method of concurrently detecting
a plurality of analytes with a single signal," or variations
thereof, refers to a method that uses a single assay (e.g. single
well, single dot, single location on an array) or a single use of a
device to detect the plurality of analytes with a single signal. If
different devices, wells, or arrays are used to detect the
plurality of analytes with the same signal this is not a method of
concurrently detecting a plurality of analytes with a single
signal. For a method to be a method of concurrently detecting a
plurality of analytes with a single signal the method must generate
only a single signal (examples of signals are described herein) in
a single location (well, dot, line on a membrane or other type of
solid support, and the like), that informs the user that the
plurality of analytes are present in the sample. For example, the
same signal being used in different wells to indicate whether a
single analyte is present in that well (or spots on an array) and
then analyzing the multiple wells (or spots) to determine if the
plurality of analytes are present is not a method of concurrently
detecting a plurality of analytes with a single signal.
[0106] As used herein, the term "single signal" means detection of
a signal based upon a single moiety or method. For example, if the
single signal is the color red, then the plurality of analytes
indicated by only upon the presence of the color red. That is, the
color red, in this non-limiting example, indicates that the
plurality analytes are present in the sample. In contrast, if one
analyte is indicated by the color red and a second analyte is
indicated by the color yellow, the use of two colors (i.e.,
signals) is not the detection of a plurality of analytes with a
single signal. The signal is not limited to colorimetric detection.
Examples are provided herein of signals that can be used.
[0107] The term "detecting" or "detection" is used in the broadest
sense to include qualitative and/or quantitative measurements of a
target analyte.
[0108] As used herein, the term "interaction unit" means a part of
the analyte or a heterologous tag or label that is attached to the
analyte that that is recognized or bound by another molecule (e.g.
the capture reagent, the bridge unit, or the signal detection
unit). The interaction unit can be part of the analyte itself or
can be a heterologous tag or label. The interaction unit can also
be an antibody or other type of capture reagent that recognizes the
analyte. In some embodiments, the analyte can comprise more 1, 2,
3, 4, or 5, interaction units. In some embodiments, the interaction
unit is a capture reagent that binds to another interaction unit
present on the analyte itself or a heterologous tag that is part of
the analyte. For example, if the analyte is a peptide or part of a
protein, a part of the protein or peptide itself can be the
interaction unit recognized by the capture reagent, bridge unit, or
the signal detection unit. In some embodiments, the peptide can
also be covalently attached to a heterologous tag or label and the
heterologous tag or label or the complex of the peptide with the
heterologous tag or label is considered the interaction unit. Thus,
in some embodiments, an analyte comprises a first interaction unit
and/or a second interaction unit. In some embodiments, the
interaction unit(s) can be intrinsic to the analyte itself or the
interaction unit(s) could be added through some other method, such
as cross-linking, covalent attachment through a chemical reaction,
non-covalent interactions (e.g. antibody-antigen, hybridization
between a part of the analyte and another molecule, and the like).
Where the interaction unit is formed by the hybridization of two
molecules (e.g. two nucleotide sequences), such that the part of
the hybridization product that is recognized by another molecule
would be considered the interaction unit. The interaction units can
also be added to the analyte through an amplification reaction.
This can be produced through the use of primers that contain the
interaction units. Interaction units can also have detectable
signals, but it is not these signals that are detected.
[0109] As used herein, the term "heterologous" in reference to the
interaction unit means a group, molecule or moiety that is not
native to the analyte. For example, an amplification product can
comprise just nucleic acid molecules or nucleotide bases. The
amplification product, however, can be conjugated to or attached to
a heterologous tag, such as, but not limited to, hapten, biotin,
digoxigenin, a fluorescent molecule (e.g. fluorescein or rhodamine)
and the like. Examples of heterologous interaction units include,
but are not limited to, hapten, biotin, nucleic acid molecules,
peptide fragments (e.g. His-tags, GST-tags, and the like), enzymes,
streptavidin, avidin, fluorescent molecules, and the like. This
list is non-limiting and any interaction unit can be used. Analytes
can be labeled with molecules such as digoxigenin, rhodamine,
fluorescein, DNP, BRDU, and then be detected by capture reagents
that are specific for a given molecule.
[0110] According to some embodiments, methods of detecting a
plurality of analytes is provided. The presently described methods
can be used to detect multiple analytes. An unexpected and
surprising result is that multiple analytes can be detected using a
single signal. This has the unexpected result that the presence, or
absence, of multiple analytes can be detected with only the
detection of one signal. This is in contrast to the detection of
the presence of multiple analytes using distinct signals in the
same reaction to detect the presence of multiple analytes in a
sample or requiring the performing of separate reactions and
methods to detect multiple analytes. That is, the embodiments
described herein provide, in part, methods of detecting multiple
analytes concurrently with a single signal, such that the detection
of a single signal indicates the presence of the multiple analytes
in a sample or that the absence of the single signal indicates the
absence of the multiple analytes in the sample. The present
embodiments provides methods of detecting at least 2, 3, 4, or 5
analytes concurrently with a single signal. In some embodiments,
the method can be used to detect 2, 3, 4, or 5 different analytes
concurrently with a single signal. Although many examples are
provided for detecting 2 analytes, the methods can be adapted and
modified based upon the present disclosure for the detection of 3,
4, or 5 analytes.
[0111] As used herein, the term "different analytes" means the
analytes are not the same. The different analytes, however, can be
referred to with the same name, but be from different organisms or
from different strains of the same organism. For example different
organisms contain genes and proteins that have the same function
and, therefore, have been given the same name. But the genes or
proteins are from different sources and thus are considered
different analytes. They may or may not have different sequences.
Different analytes can also means analytes from different
organisms. For example, there are any many strains of E. coli. Not
all strains of E. coli cause a food-borne illness. The present
methods can be used, for example, to detect a plurality of analytes
from a pathogenic E. coli strain as opposed to detecting an analyte
from a non-pathogenic E. coli strain. Although reference made be
made throughout the present disclosure to specific types of
analytes, the analytes can be any type of analyte, such as but not
limited, to the classes of analytes described herein.
[0112] For example, in some embodiments, methods of concurrently
detecting a first analyte and a second analyte are provided. In
some embodiments, the method comprises contacting a solid support,
which comprises a first capture reagent with a first analyte, a
second analyte, a bridge unit, which comprises a second capture
reagent, and a signal detection unit comprising a third capture
reagent; and detecting the presence of the signal detection unit
which indicates the presence of the first analyte and second
analyte. In some embodiments, the first capture reagent is affixed
to the solid support. In some embodiments, the first analyte
comprises a first interaction unit that binds to the first capture
reagent and a second interaction unit that binds to the bridge
unit; the second analyte comprises a first interaction unit that
binds the bridge unit and a second interaction unit that binds to
the signal detection unit. The signal detection unit can then be
detected. If the signal detection unit is detected, it indicates
that the multiple analytes are present.
[0113] Without desiring to be bound by any theory, the multiple
analytes can be detected concurrently by forming a complex. In some
embodiments, the complex comprises the solid support, the first
analyte, the second analyte, the bridge unit, and the signal
detection unit wherein each member of the complex binds to each
other directly or indirectly. The sample can be washed while
retaining the solid support and the complex will only be detected
if the complex is formed. Examples of these complexes can be seen
in FIGS. 1-3, which are further described herein.
[0114] In some embodiments, methods of concurrently detecting a
first analyte and a second analyte are provided, the method
comprising: contacting a solid support with a first analyte of
interest, a second analyte of interest, a bridge unit comprising a
second capture reagent, and a signal detection unit comprising a
third capture reagent; and detecting the presence or absence of the
signal detection unit which indicates the presence or absence of
the first analyte of interest and second analyte of interest
concurrently, wherein: a first capture reagent is affixed to the
solid support; the first analyte of interest comprises a first
interaction unit that binds to the first capture reagent and a
second interaction unit that binds to the bridge unit; and the
second analyte of interest comprises a first interaction unit that
binds the bridge unit; a signal detection unit that binds to the
second analyte, to the second analyte's first interaction unit or a
second interaction unit, to a component of the first and second
analyte complex or bridge unit that that is only present when the
complex contains the first and second analyte.
[0115] FIG. 1 illustrates a complex that could be formed to detect
two analytes concurrently with a single signal. FIG. 1 illustrates
a capture reagent 15 affixed to a solid support 10. The capture
reagent 15 binds to the first analyte 20. The bridge unit 30 binds
to the first analyte. The bridge unit also binds to the second
analyte 40. FIG. 1 also illustrates a signal detection unit 60
comprising a capture reagent 50 that binds to the second analyte
40. FIG. 1 illustrates that this complex only forms when all of the
members are present and can bind to one another. The signal
detection unit can then be detected. FIG. 3 also shows an
embodiment of detecting two analytes with a single signal. FIG. 3
shows specific labels (e.g. FITC, TAMRA, DIG, biotin, streptavidin,
etc . . . ), but these labels can be modified according to the
present disclosure.
[0116] In some embodiments, the method comprises one or more
washing steps. The washing step can be used to remove unbound
materials. For example, if the solid support is contacted with a
first sample, the solid support can be washed to remove any unbound
material. In some embodiments where the solid support is a bead,
the beads can be contacted with the sample and then the beads can
be washed. Washing beads is routine and well known to one of skill
in the art. The method of washing beads or other types of solid
supports can be altered or chosen based upon the specific solid
support that is used and is often not a critical feature.
[0117] In some embodiments, the sample with the first analyte is
contacted with the solid support. In some embodiments, the mixture
is washed such that any materials not bound to the solid support
are no longer present. In some embodiments, the solid support is
also contacted with the same sample or a different sample
comprising the second analyte and/or the bridge unit. The mixture
can then be washed again to remove any unbound material. In some
embodiments, a signal detection unit comprising a capture reagent
is added. A washing step can also be included to remove any unbound
signal detection units. The signal detection unit can then be
detected or another reagent can be added that detects the presence
of the signal detection unit. In some embodiments, all of the steps
are performed simultaneously or nearly simultaneously. During the
performance of the method, a washing step may be inserted where
appropriate.
[0118] In some embodiments, the different analytes or samples can
be mixed together before or simultaneously applied to the solid
support. The samples can be, for example, amplification reaction
mixtures that were used to produce, or attempted to produce, the
analytes. In some embodiments, different amplification reactions
will be performed to amplify the plurality of analytes. Therefore,
prior to the samples or analytes being applied to the solid support
the samples or analytes can be mixed together. The samples or
analytes can also be mixed with the capture reagents and/or
bridging units prior to being contacted with the solid surface.
[0119] In some embodiments, the first and second interaction unit
of the first analyte and the first and second interaction unit of
the second analyte are each independently a heterologous
interaction unit. In some embodiments, an interaction unit of the
first analyte and an interaction unit of the second analyte is a
hapten. In some embodiments, the interaction unit of the first
analyte and the second analyte is fluorescein or rhodamine
molecule. Accordingly, in some embodiments, the first analyte and
second analyte have at least one interaction unit in common. The
commonality of the interaction unit will enable the bridging unit
to bring the two analytes into a detectable complex. In some
embodiments, the first and second analytes do not have the same
interaction unit. In such a case for some embodiments, the bridging
entity would be a bivalent capture reagent (e.g. bivalent antibody)
that can link the two analytes to one another. The bivalent capture
reagent would be able to bind to both the first and second analytes
simultaneously. In some embodiments, each of the interaction units
present on the plurality of analytes are different. In some
embodiments, some of the interactions units are different, but some
of the interaction units are the same. In some embodiments, the
analyte comprises a hapten interaction unit and a biotin
interaction unit. In some embodiments, the first analyte comprises
a digoxigenin interaction unit and a rhodamine interaction unit;
the second analyte comprises a rhodamine interaction unit and a
FITC interaction unit, and the bridging unit binds to the rhodamine
interaction unit. The bridging unit can then form the complex that
contains both the first and second analyte. This can be seen, for
example, in FIG. 3.
[0120] In some embodiments, the plurality of the analytes are the
same type of analyte. For example, each of the analytes being
detected can be a peptide. In some embodiments, each of the
analytes is a nucleic acid molecule, such as an amplification
product (e.g. amplicon). The analytes can also be any type,
including, but not limited to, the analytes described herein. In
some embodiments, the analytes are different. In some embodiments,
a first analyte is an amplification product and a second analyte is
a protein or peptide. Any combination of analytes can be used.
[0121] As used herein, the term "bridge unit" or "bridge" means a
molecule(s) that can link two or more analytes in a complex. That
is, for example, the bridge unit can bind to an interaction unit on
a first analyte and an interaction unit on a second analyte. If
only detecting two analytes, one bridge unit may be used. If
detecting three analytes, two bridge units can be used. In some
embodiments, the methods use "n-1" bridge units, where "n" is the
number of analytes being detected. In some embodiments, a single
bridge unit is used to detect more than 2 analytes. Examples of
bridge units include, but are not limited to, immunoglobulin
molecules (e.g. IgM, IgE, IgG, IgA, and the like), streptavidin,
and a molecule that comprises a plurality of capture reagents such
that the bridge unit can bind to more than one interaction unit. In
some embodiments, the bridge unit is a multivalent capture
reagent.
[0122] In some embodiments, the bridge unit is a complex of
compounds, substances, or macromolecules. For example, a bridge
unit could comprise a nanoparticle coated with antibodies and a
separate antibody. In this non-limiting example, the nanoparticle
coated with antibodies can contain antibodies that bind to an
analyte or interaction unit on the analyte and also contain
antibodies that bind to the separate antibody. The separate
antibody can bind to a different analyte. The interaction of the
nanoparticle coated with antibodies and the separate antibody would
then be able to bridge together the different analytes. A
non-limiting illustration of this bridge complex can be seen in
FIGS. 4 and 5, which is also described below. Other variations of
the bridge being a complex could also be made. The exact structure
and form of the bridge unit is not essential so long as it can
"bridge" a plurality of analytes in a complex. Thus, the bridge
could be made up of multiple subunits or components to bridge the
analytes together. Although the bridge unit can be illustrated and
discussed bridging two analytes, the bridge unit can be designed to
bridge more than 2 analytes, such as 3, 4, 5, or more. Therefore,
in some embodiments, the bridge unit bridges 2, 3, 4, 5, or more
analytes. In some embodiments, the bridge unit bridges at least 2,
3, 4, or 5 analytes. The non-limiting example of bridging 2
analytes is for illustrative purposes only and the embodiments
disclosed herein are not limited to bridging only 2 analytes.
[0123] As discussed herein, the present methods can be applied to
detecting more than 2 analytes. For example, a method of detecting
a first analyte, a second analyte, and a third analyte concurrently
with a single signal is provided. For the detection of additional
analytes, the methods can be adapted in a similar manner. In some
embodiments, the method comprises contacting the first, second, and
third analytes with a solid support, a first bridge unit, a second
bridge unit, and a signal detection unit; and detecting the
presence of the signal detection unit which indicates the presence
of the first, second, and third analytes concurrently with a single
signal, wherein the first analyte comprises a first interaction
unit and a second interaction unit; the second analyte comprises a
first interaction unit and a second interaction unit; the third
analyte comprises a first interaction unit and a second interaction
unit; the solid support comprises a first capture reagent that
binds to the first interaction unit of the first analyte; the first
bridge unit binds to the second interaction unit of the first
analyte and the first interaction unit of the second analyte; the
second bridge unit binds to the second interaction unit of the
second analyte label and the third interaction unit of the third
analyte; and the signal detection unit binds to the second
interaction unit of the third analyte. Without being bound to any
theory, it is expected that the analytes can be concurrently
detected because the first, second, and third analytes form a
complex, wherein the complex comprises the solid support, the first
analyte, the second analyte, the third analyte, the first bridge
unit, the second bridge unit, and the signal detection unit wherein
each member of the complex binds to each other directly or
indirectly.
[0124] FIG. 2 illustrates a complex that can be formed to detect
three analytes, which is analogous to the example illustrated in
FIG. 1. FIG. 2 illustrates a solid support 10 with a capture
reagent 15 bound to a first analyte 70. The first analyte is bound
to a first bridge unit 80. The first bridge unit is bound to a
second analyte 20, which is also bound to a second bridge unit 30.
The second bridge unit is also bound to a third analyte 40, which
is bound to a capture reagent 50. The capture reagent is also
attached to a signal detection unit 60. Thus, FIG. 2 illustrates a
non-limiting example of how three analytes can be detected with a
single signal.
[0125] FIG. 4 illustrates a non-limiting bridge complex made up of
more than one molecule, macromolecule, or substance. This can be
referred to as a multi-component bridge complex. FIG. 4 illustrates
a bridge unit 30 that comprises a particle 34, a first capture
reagent 31, a second capture reagent 32, and a third capture
reagent 33. The bridge unit 30 is able to bring together the first
analyte 20 and the second analyte 40 and from a complex linking the
first analyte 20 and the second analyte 40. FIG. 4 illustrates a
particle 34 (e.g. nanoparticle, polystyrene, agarose, and the like)
coated with a first capture reagent 31 that binds to the first
analyte 20, either directly or indirectly through an interaction
unit, a third capture reagent 33, which is also present on the
particle 34, that binds to a second capture reagent 32 that is
bound to the second analyte 40. This complex can then be detected
according to the methods and compositions described herein, which
is illustrated in FIG. 5. FIG. 5 shows the bridges complex of FIG.
4 interacting with a solid support 10 with a capture reagent 15
bound to a first analyte 20 and a signal detection unit 60
comprising a capture reagent 50 that binds to the second analyte
40. As discussed herein, the illustration of the signal detection
unit binding to the second analyte is for illustrative purposes
only. The signal detection unit can also bind other parts of the
complex so long as the signal detection unit is not binding to the
analyte that is interacting with the solid support or the solid
support itself. The solid support 40 can be, for example, a test
membrane, such as the test membrane that is shown in FIG. 3. Other
examples of solid supports are provided herein.
[0126] The present invention provides complexes comprising a solid
support, a first analyte, a second analyte, a bridge unit, and a
signal detection unit wherein each member of the complex binds to
each other directly or indirectly. In some embodiments, the solid
support is bound to the first analyte, the bridge unit is bound to
the first analyte and the second analyte, and the signal detection
unit is bound to the second analyte. In some embodiments, the solid
support comprises a first capture reagent, the first analyte
comprises a first interaction unit and a second interaction unit,
the second analyte comprises a first interaction unit and a second
interaction unit, the bridge unit comprises one or more capture
reagents that independently bind to the second interaction unit of
the first analyte and the first interaction unit of the second
analyte, and the signal detection unit comprises a capture reagent
that binds to the second interaction unit of the second
analyte.
[0127] In some embodiments, the complex comprises a solid support,
a first analyte, a second analyte, a third analyte, a first bridge
unit, a second bridge unit, and a signal detection unit, wherein
the solid support, the first analyte, second analyte, third
analyte, first bridge unit, second bridge unit, and signal
detection unit are bound to each other directly or indirectly. In
some embodiments, the solid support binds to the first analyte, the
first bridge unit binds to the first analyte and the second
analyte, the second bridge unit binds to the second analyte and the
third analyte, and the signal detection unit binds to the third
analyte. In some embodiments, the solid support comprises a first
capture reagent, the first analyte comprises a first interaction
unit and a second interaction unit, the second analyte comprises a
first interaction unit and a second interaction unit, the third
analyte comprises a first interaction unit and a second interaction
unit, the first bridge unit comprises one or more capture reagents
that independently bind to the second interaction unit of the first
analyte and the first interaction unit of the second analyte, the
second bridge unit comprises one or more capture reagents that
independently bind to the second interaction unit of the second
analyte and the first interaction unit of the third analyte, and
the signal detection unit comprises a capture reagent that binds to
the second interaction unit of the third analyte.
[0128] In some embodiments, the presently described methods can be
used to detect a food borne pathogen by the detection of a
plurality of food-borne pathogen analytes with a single signal. For
example, a sample may be processed to isolate an analyte (e.g. an
antigen or a toxin, or a food borne pathogen nucleic acid may be
isolated or amplified). The plurality of analytes (e.g. food borne
pathogen protein and/or an amplicon) can be detected concurrently
with the methods described herein. The methods can then provide
greater confidence in the specificity of the test and avoid false
negatives. In some embodiments, a positive result that indicates
the presence of a food borne pathogen requires the detection of 2,
3, or 4 analytes. The present methods can be used to detect the
analytes concurrently with a single signal. The single signal
provides an easier result to interpret since the signal will only
be detectable if all of the plurality of analytes being detected
are present in the sample. Thus, if 2 analytes are being detected
then the signal will only be detectable if both analytes are
present. In some embodiments, the signal is only detectable when 3
analytes are present. This type of methods can be applied to other
methods of detection.
[0129] In some embodiments, the method can be used to detect 3
classes of analytes to provide a positive test for food
contamination. In some embodiments, one of the analytes is a toxin
(e.g. Shiga toxin 1 and/or 2). The toxin can be detected itself or
the nucleotide sequence that encodes or controls the production of
the toxin can be detected. In some embodiments, one of the analytes
is eae gene, which can also be referred to as a virulence factor.
The eae gene can be found, or expressed in, for example,
enterohemorrhagic Escherichia coli.
[0130] In some embodiments one of the analytes is a serotype
analyte, which can be an antigen that is specifically produced by a
strain of a food borne pathogen. In some embodiments, the serotype
analyte is an E. coli serotype. In some embodiments, the E. coli
serotype is 026, 045, 0103, 0111, 0121, and 0145. Therefore, in
some embodiments, a positive test for food borne contamination
requires the detection of 3 analytes with a single signal, wherein
the 3 analytes are the Shiga toxin (e.g. Shiga toxin 1 and/or 2),
the eae gene, and a serotype analyte chosen from E. coli serotype
is 026, 045, 0103, 0111, 0121, and 0145. In some embodiments, the
serotype analyte is a protein specifically expressed by a
pathogenic strain. In some embodiments, the analyte is a nucleic
acid sequence that encodes the antigen. In some embodiments, the
nucleic acid sequence is a fragment of the coding sequence of the
antigen. The specific fragment is not critical and one of skill in
the art can determine the sequences or fragments thereof to amplify
using routine methods. As discussed herein, the target sequence can
be amplified and optionally labeled with a heterologous interaction
unit. The analytes can then be detected according to the methods
provided herein.
[0131] For example, if a positive test for a virus requires the
presence of two distinct nucleic acid sequences, the two nucleic
acid sequences can be detected concurrently with a single signal
using the methods described herein as opposed to detecting the two
nucleic acid sequences separately with more than one signal.
Additionally, if the presence of cancer requires the detection of a
plurality of genes being expressed in sample, the genes can be
detected concurrently with a single signal by using analytes that
correlate with their expression (e.g. by using RT-PCR to amplify
the gene product) according to a method described herein.
Therefore, the presently described methods have wide applicability
and can be used with any plurality of analytes (target molecules)
and even with analytes that are not the same.
[0132] In some embodiments, methods are provided for detecting two
or more analytes comprising detecting the multiple analytes using a
flow (vertical or lateral) device. Examples of vertical flow
devices and methods of using them are provided in U.S. Pat. Nos.
8,012,770, 8,183,059 and U.S. patent application Ser. No.
13/500,997, Ser. No. 13/360,528, Ser. No. 13/445,233, each of which
is hereby incorporated by reference in its entirety. The devices
can be adapted for the detection of multiple analytes using a
single signal.
[0133] Accordingly, embodiments provided herein provide methods of
detecting multiple analytes with a single signal by using vertical
flow and devices employing vertical flow. Vertical flow allows the
analyte and/or the sample to flow through the layers/membranes of
the analyte detection membrane system. By "through layers" or
"through membranes" is meant to refer to the sample flowing through
the layers and vertically across the layers. In some embodiments,
the sample does not flow horizontally or laterally across the
different layers/membranes.
[0134] The following terms are used in conjunction with the
description of various vertical flow devices. Other terms are
defined relevant to some vertical flow devices or uses thereof are
described throughout as well.
[0135] The term "pressure actuator" and "force actuator" can be
used interchangeably and refer to a component that can exert, for
example, pressure through the application of force. A force
actuator can also be referred to as a force member. Examples of
include, but are not limited to, various force members that are
described herein. Other examples include, but are not limited to,
pistons or other solid support structures. The force actuator's
position relative to another component can be raised, lowered, or
moved laterally. The position of the force actuator can be
controlled manually or through a signal processing unit (e.g.
computer). The ability to control the position of the force
actuator can be used to regulate the force (e.g. pressure) being
applied to another component, such as, but not limited to, an
analyte detection membrane system. By regulating the force applied
to the membrane system the flow rate of the sample can be
regulated. The force can be used to keep the flow rate of the
sample through the membrane system constant or the flow rate can be
variable. The flow rate can also be stopped and allow the sample to
dwell on different layers of the membrane system. For example, the
sample's flow rate can be zero or near zero when the sample
contacts the conjugate pad. After resting on the conjugate pad the
flow rate can be increased by modulating the pressure being applied
by the force actuator. The sample can then through the entire
membrane system, or the force being applied can be modulated to
allow the sample to dwell (rest) on another layer of the membrane
system. Force can be precisely regulated, either manually or by
using a signal processing unit (e.g. computer) the flow rate can be
modified at any point as the sample vertically flows through the
membrane system. The flow rate can also be regulated based upon the
absorbency of the membranes in the membrane system and/or the
number of the membranes of the system. Based upon the absorbency
the flow rate can be modulated (e.g. increased or decreased).
[0136] The flow rate can be measured in any units including but not
limited to .mu.l/min or .mu.l/sec, and the like. The flow rate
during a dwell can be, for example, 0 .mu.l/sec, or less than 1,
0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 .mu.l/sec or
.mu.l/min. The flow rate can be monitored manually or by a signal
processing unit (e.g. computer) and regulated by the same. The flow
rate can be regulated and monitored by well-known and routine
methods known to one of skill in the art in addition to those
described herein. In some embodiments, the flow rate is about 0 to
1 ml/min, about 0-10 ml/min, about 1-9 ml/min, about 1-8 ml/min,
about 1-7 ml/min, about 1-6 ml/min, about 1-5 ml/min, about 1-4
ml/min, about 1-3 ml/min, about 1-2 ml/min, about 0.5-1.5 ml/min,
about 1-1.5 ml/min, or about 0.5-1 ml/min. In some embodiments, the
flow rate is about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ml/min. In some
embodiments, the flow rate is at least 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10 ml/min. In some embodiments, the flow rate is 1, 2, 3, 4, 5,
6, 7, 8, 9, or 10 ml/min.
[0137] In some embodiments, the devices used to detect multiple
analytes with a single signal comprise a housing comprising a first
housing member and a second housing member. In some embodiments,
the first and second housing members can be constructed as a single
unit. The housing can comprise an inlet opening. The inlet opening
allows the introduction of a sample onto the chromatographic assay.
In some embodiments, the first housing member comprises the inlet
opening. The inlet opening can be of sufficient size to handle an
appropriate amount of volume of a solution that is added to the
device. In some embodiments, the size of the opening is large
enough to handle about 0.1 to 3 ml, about 0.1 to 2.5 ml, about 0.5
to 2.0 ml, about 0.1 to 1.0 ml, about 0.5 to 1.5 ml, 0.5 to 1.0 ml,
and 1.0 to 2.0 ml.
[0138] In some embodiments, the housing comprises a conjugate pad,
a permeable membrane, a test membrane, and/or an absorbent member.
In some embodiments, the housing comprises an analyte detection
membrane system. In some embodiments, the analyte detection
membrane system comprises a conjugate pad, a permeable membrane, a
test membrane, and an absorbent member. In some embodiments, the
analyte detection membrane system is free of a permeable membrane.
In some embodiments, the analyte detection membrane system
comprises in the following order: a conjugate pad, a permeable
membrane, a test membrane, and an absorbent member.
[0139] As used herein, the term "conjugate pad" refers to a
membrane or other type of material that can comprise a capture
reagent. The conjugate pad can be a cellulose acetate, cellulose
nitrate, polyamide, polycarbonate, glass fiber, membrane,
polyethersulfone, regenerated cellulose (RC),
polytetra-fluorethylene, (PTFE), Polyester (e.g. Polyethylene
Terephthalate), Polycarbonate (e.g.,
4,4-hydroxy-diphenyl-2,2'-propane), Aluminum Oxide, Mixed Cellulose
Ester (e.g., mixture of cellulose acetate and cellulose nitrate),
Nylon (e.g., Polyamide, Hexamethylene-diamine, and Nylon 66),
Polypropylene, PVDF, High Density Polyethylene (HDPE) +nucleating
agent "aluminum dibenzoate" (DBS) (e.g. 80 u 0.024 HDPE DBS
(Porex)), and HDPE. Examples of conjugate pads also include,
Cyclopore.RTM. (Polyethylene terephthalate), Nucleopore.RTM.
(Polyethylene terephthalate), Membra-Fil.RTM. (Cellulose Acetate
and Nitrate), Whatman.RTM. (Cellulose Acetate and Nitrate), Whatman
#12-S (rayon)), Anopore.RTM. (Aluminum Oxide), Anodise.RTM.
(Aluminum Oxide), Sartorius (cellulose acetate, e.g. 5 .mu.m), and
Whatman Standard 17 (bound glass). The conjugate pad can also be
made of a material that dissolves after coming into contact with a
sample or other liquid. The dissolving of the conjugate pad can be
performed so that other layers of the systems described herein can
be revealed or exposed for either visual inspection (e.g. detection
of an analyte) or for spectrometer inspection (e.g. detection of an
analyte by a spectrometer).
[0140] In some embodiments, the conjugate pad or test membrane
comprises a capture reagent. In some embodiments, the conjugate pad
or test membrane is contacted with the capture reagent and then
allowed to dry before being used in the vertical flow device. The
conjugate pad or test membrane can also comprise other compositions
to preserve the capture reagent such that it can be stably stored
at room temperature or under refrigeration or freezing
temperatures. In some embodiments, the conjugate pad or test
membrane is soaked with a buffer prior to the capture reagent being
applied. In some embodiments, the buffer is a blocking buffer that
is used to prevent non-specific binding. In some embodiments, the
buffer comprises Borate, BSA, PVP40 and/or Tween-100, or any
mixture thereof. In some embodiments, the buffer is 10 mM Borate,
3% BSA, 1% PVP40, and 0.25% Tween-100. In some embodiments the
capture reagent is applied to the pad or membrane in a solution
comprising trehalose and sucrose. In some embodiments, the capture
reagent is applied to the pad, membrane, or both, in a solution
comprising trehalose, sucrose and phosphate and/or BSA. In some
embodiments, the capture reagent is applied in a solution that is
5% trehalose, 20% sucrose, 10 mM phosphate, and 1% BSA. In some
embodiments, the test membrane comprises a capture reagent that
binds to a labeled amplicon. In some embodiments, the capture
reagent is an antibody that recognizes or binds to digoxigenin,
fluorescein (e.g. FITC), rhodamine (TAMRA), and the like.
[0141] In some embodiments, the conjugate pad comprises
streptavidin. The streptavidin can also be further labeled as
described herein. In some embodiments, the streptavidin is the
capture reagent that binds to a biotinylated antibody that is used
to detect multiple analytes with a single signal.
[0142] In some embodiments, the removable member contacts a first
surface of the conjugate pad and the adhesive member contacts a
second surface of the conjugate pad.
[0143] In some embodiments, the device comprises an adhesive
member. The adhesive member can comprises an adhesive member inlet
that allows the sample to flow through the conjugate pad and
contact the test membrane. In some embodiments, the adhesive member
inlet is the same size or shape as the removable member inlet. In
some embodiments, the adhesive member inlet is a different size or
shape as the removable member inlet. In some embodiments, the
inlets in the adhesive member are the same shape but have different
areas. Inlets with different areas would be considered to have
different sizes. The adhesive member can be made up of any
substance suitable for adhering one member or membrane to another
member or membrane. In some embodiments, the adhesive member is
impermeable to liquid. In some embodiments, the adhesive member
contacts the removable member.
[0144] In some embodiments of the device, the permeable membrane is
attached to or adhered to a test membrane. In some embodiments, the
permeable membrane is laminated onto the test membrane. The
permeable membrane can be a membrane of any material that allows a
sample, such as a fluid sample, to flow through to the test
membrane. Examples of test membrane include, but are not limited
to, nitrocellulose, cellulose, glass fiber, polyester,
polypropylene, nylon, and the like. In some embodiments, the
permeable membrane comprises an opening. The opening can be present
to allow visualization or detection of the test membrane. In some
embodiments, the opening in the permeable membrane is substantially
the same size as the inlet opening in the housing. Examples of
permeable membranes include, but are not limited to, Protran BA83,
Whatman, and the like.
[0145] As discussed herein, one example of a solid support is a
test membrane. As used herein and throughout, the "test membrane"
refers to a membrane where detection of a binding partner to a
capture reagent occurs. Test membranes include, but are not limited
to a nitrocellulose membrane, a nylon membrane, a polyvinylidene
fluoride membrane, a polyethersulfone membrane, and the like. The
test membrane can be any material that can be used by one of skill
in the art to detect the presence of a capture reagent's binding
partner (e.g. labeled analyte, antigen or epitope). The test
membrane can also comprise a capture reagent. In some embodiments,
the test membrane is contacted with a capture reagent and the
capture reagent is allowed to dry and adhere to the test membrane.
Examples of test membranes include, but are not limited to Protran
BA83, Whatman, Opitran BA-SA83, and 0.22 .mu.m white plain
(Millipore Product No. SA3J036107). Test membranes may also be
comprised of nanoparticle matrices to which capture reagents are
bound. Nanocrystals can be arranged into 2D sheets and 3D matrices
with materials such as, but not limited to, carbon based particles,
gold or metal alloy particles, co-polymer matrices, as well as
monodisperse semiconducting, magnetic, metallic and ferroelectric
nanocrystals. The test membrane can comprise a plurality of capture
reagents. In some embodiments, the test membrane comprises 1, 2, 3,
4, 5, 6, 7, 8, 9, or 10 capture reagents. In some embodiments, the
test membrane comprises a plurality of areas each with a different
capture reagent. In some embodiments, the plurality of areas do not
completely overlap or coincide with one another.
[0146] In some embodiments, the device or housing also comprises an
absorbent member. The absorbent member can also be referred to as a
"wick pad" or "wicking pad." The absorbent member absorbs the fluid
that flows through the device when the sample is applied to the
device and provides for the wicking force that aids in the flow of
the sample when it is applied to the device. "Absorbent member" is
meant to refer to a material that has a capacity to draw (wick) and
retain solution away from a surface that the material is in contact
with. Use of a combination of material of increasing or decreasing
absorbance can also allow for control of sample movement.
[0147] The absorbent member can be any material that can facilitate
the flow of the sample through the conjugate pad and to the test
membrane. Examples of absorbent members include, but are not
limited to cellulose, super absorbent polymers, glass fiber pads
(e.g. C083 (Millipore)), and the like. In some embodiments, the
housing comprises a plurality (e.g. 2 or more) of absorbent
members. In some embodiments, the housing comprises 2, 3, 4, or 5
absorbent members. In some embodiments, the device comprises one
absorbent member. In some embodiments, the absorbent member
comprises one or more membranes up to 10 individual membranes, and
each membrane may be the same material or a different material. In
some embodiments, the device consists of only 1 membrane that is an
absorbent member.
[0148] In some embodiments, the device comprises a force member.
Examples of force members are described below and can be seen in
the drawings. These examples are non-limiting and other forms of
force members can be used. The force member can, in some
embodiments, be used to apply pressure or to compress the other
components of the analyte detection membrane system against one
another. In some embodiments, the force member can be made out of
any material including, but not limited to plastic or stainless
steel. As shown in FIG. 23, clips can act as force members. The
stainless steel can be laser cut such that it can act as a clip.
Non-limiting examples of these clips can be seen in FIG. 23. The
force member acts to apply pressure to the membrane system. The
force member is not limited to a clip, but rather can be any shape
(see, Figures for non-limiting examples) that can apply pressure to
the membrane system (e.g. nanoparticle matrices) and piston like
structures strategically placed within the assembly. In some
embodiments, the force member is a piston. The force member can be
used to apply pressure or to compress the other components of the
analyte detection membrane system against one another. In some
embodiments, the force member can comprise a shaft and a head. The
force member can have a mushroom type shape where the head is wider
than the shaft. In some embodiments, the head is narrower than the
shaft. The force member comprising a head and a shaft can be a
single unit or can be made up of multiple parts that contact one
another to form the force member. For example, the head could be
one unit that can be separated from the shaft. Upon assembly the
head and shaft are contacted with one another to make the force
member. In another example, the head and shaft are one cohesive
unit and are manufactured together and not as separate parts that
are later assembled to form the force member. The force member
allows the device to work with vertical flow as opposed to relying
upon lateral flow.
[0149] In some embodiments, the force member contacts a surface of
the absorbent member. In some embodiments, the force member
contacts a surface of the absorbent member and a surface of the
removable layer. In some embodiments, the force member compresses
the membrane detection system from above and below the membrane
detection system. For example, in some embodiments, the force
member can sandwich all the layers of the membrane detection
system. In some embodiments the force member is attached to a
support member.
[0150] In some embodiments, the device comprises, in the following
order, a removable member, a conjugate pad, and an adhesive
member.
[0151] The device can also comprise a support member. The support
member, in some embodiments, contacts a surface of the absorbent
member. The support member can also have a support member inlet.
The inlet can be the same size and/or shape as the inlet in the
removable member and/or the adhesive member. In some embodiments,
the support member comprises an inlet that is a different size
and/or shape as the inlet in the removable member and/or the
adhesive member. The support member can be made from any material
including, but not limited to, plastic. In some embodiments, the
second housing member serves as the support member.
[0152] The devices described herein can be used in assays to detect
the presence of a capture reagent's binding partner. These assays
can as shown herein be used to detect multiple analytes for the
detection of single signals. For example, an antigen can be
detected by an antibody using the devices of the present invention.
The term "Vertical flow" is used throughout. The term "vertical
flow" refers to the direction that the sample flows across the
different membranes and members present in a device. Vertical flow
refers to a sample flowing through the membrane (e.g. top to
bottom) as opposed to lateral flow, which refers to a sample
flowing across (e.g. side to side) a membrane, pad or absorbent
member. In a lateral flow device the membranes and pads sit
horizontally adjacent to one another substantially on the same
plane. In a vertical flow device each membrane or pad is
substantially parallel or completely parallel to each other and
occupy substantially different spatial planes in the device. The
membranes and pads may occupy similar planes when they are
compressed or put under pressure. In some embodiments, at least a
portion of each member, membrane, or pad is layered on top of each
other. In some embodiments, at least a portion of each layer of
member, membrane, or pad is substantially parallel to each other.
In some embodiments, at least a portion of each layer is in a
different spatial plane than each other layer.
[0153] To allow vertical flow to occur efficiently, in some
embodiments and when present, the conjugate pad, permeable
membrane, test membrane and the absorbent member are substantially
parallel to each other. In some embodiments, the conjugate pad,
permeable membrane, test membrane and the absorbent member are
present in different spatial planes. In some embodiments, the
housing also comprises a hydrophobic membrane that can slow or stop
the vertical flow of the sample. The hydrophobic membrane can be in
contact with the test membrane, which would allow the sample to
dwell or rest upon the test membrane. The dwell can allow for
increased sensitivity and detection. The vertical flow is modulated
by the pressure that is applied to the membranes, pads, and/or
members. In some embodiments, the pressure is applied perpendicular
to the test membrane and/or the conjugate pad. In some embodiments,
the pressure can be applied so that the conjugate pad is compressed
against the housing. The compression against the housing can be
such that the conjugate is in direct contact with the housing,
O-ring, or collar, or through an intermediate so that the conjugate
pad and the test membrane are compressed against one another.
[0154] The force member can apply pressure that is substantially
perpendicular to the test membrane. Without being bound to any
particular theory, the pressure facilitates the vertical flow. The
pressure allows each layer of the membrane stack to be in contact
with another layer. The pressure can also be relieved to stop the
flow so that the test sample can dwell or rest upon the test
membrane, which can allow for greater sensitivity. The pressure can
then be reapplied to allow the vertical flow to continue by
allowing the sample to flow into the absorbent member(s). The force
member can apply pressure such that the conjugate pad contacts a
portion of the housing (e.g., first or second housing members or
removable layer). In some embodiments, the conjugate pad contacts
the housing when it is not under the pressure being exerted by the
force member but upon the force member exerting pressure the
conjugate pad is compressed against a portion of the housing.
[0155] In some embodiments, the conjugate pad contacts the
perimeter of the inlet opening. The inlet opening can also comprise
a collar or other similar feature, such as an O-ring. In some
embodiments, the conjugate pad contacts the perimeter of a collar
and/or an O-ring. In some embodiments, the conjugate pad is capable
of being compressed against the perimeter of the inlet opening,
which can include, in some embodiments, a collar and/or an
O-ring.
[0156] "Capable of being compressed against the perimeter of the
inlet opening" refers to a membrane or pad (e.g. conjugate pad)
being compressed either directly in contact with the perimeter of
the inlet opening or being compressed against another layer or
material (e.g. membrane) that is in contact with the perimeter of
the inlet opening.
[0157] In some embodiments, the conjugate pad is not in direct
physical contact with the housing but is in fluid contact with the
housing. "Fluid Contact" means that if a sample is applied to the
device through the inlet opening or other opening the fluid will
contact the conjugate pad. In some embodiments, the conjugate pad
can be separated from the housing by another membrane, such as a
permeable membrane, where the other membrane is in direct physical
contact with the housing or in direct physical contact with the
collar or O-ring. When the sample is applied to the device the
fluid can contact the other membrane first and then contact the
conjugate pad. This is just one example of the conjugate pad being
in fluid contact with the housing. There are numerous other
embodiments where the conjugate pad is not in direct physical
contact with the housing, the collar, or the O-ring, but is in
fluid contact with the housing.
[0158] The force member can apply any pressure that is sufficient
to facilitate vertical flow across the different membrane layers.
In some embodiments, the layers of the device (e.g. conjugate pad,
permeable membrane, test membrane, and absorbent member) are
compressed under a force chosen from about 5 lbf to 100 lbf, about
5 lbf to 50 lbf, about 10 lbf to 401 bf, about 15 lbf to 40 lbf,
about 15 lbf to 25 lbf, or about 30 lbf to 40 lbf. In some
embodiments, the layers of the device (e.g. conjugate pad,
permeable membrane, test membrane, and absorbent member) are
compressed under a force chosen from about 1 lbf to 100 lbf, about
1 lbf to 50 lbf, about 1 lbf to 5 lbf , about 1 lbf to 10 lbf,
about 1 lbf to 15 lbf, about 1 lbf to 20 lbf, about 1 lbf to 30
lbf, or about 1 lbf to 25 lbf. The force can also compress a
hydrophobic or impermeable membrane as well if one is present in
the device.
[0159] In some embodiments of a device that can be used to detect
multiple analytes with a single signal, the force member contacts a
first surface of an absorbent member. In some embodiments, a
conjugate pad contacts a test membrane. In some embodiments, a
first surface of a test membrane contacts a permeable membrane. In
some embodiments, a second surface of the test membrane contacts a
second surface of the absorbent pad. In some embodiments, the
device comprises a hydrophobic membrane, and, for example, the
hydrophobic membrane contact a second surface of the test membrane.
In some embodiments, the hydrophobic membrane contacts a first
surface of the absorbent pad. In some embodiments, a conjugate pad
contacts an adhesive member. In some embodiments, a test membrane
contacts an adhesive member.
[0160] In some embodiments of a device that can be used to detect
multiple analytes with a single signal, a first surface of the
conjugate pad contacts the housing and a second surface of the
conjugate pad contacts a first surface of the permeable membrane,
wherein the second surface of the permeable membrane contacts a
first surface of the test membrane, wherein a second surface of the
test membrane contacts a first surface of the absorbent pad,
wherein a second surface of the absorbent pad contacts the force
member. In some embodiments, the first surface of the conjugate pad
contacts a perimeter of the inlet opening of said housing. In some
embodiments, the first surface of the conjugate pad contacts a
perimeter of a collar or an O-ring.
[0161] In some embodiments of a device that can be used to detect
multiple analytes with a single signal, a first surface of the
conjugate pad contacts the housing and a second surface of the
conjugate pad contacts a first surface of the adhesive member,
wherein the second surface of the adhesive member contacts a first
surface of the test membrane, wherein a second surface of the test
membrane contacts a first surface of the absorbent pad, wherein a
second surface of the absorbent pad contacts the support member. In
some embodiments, the first surface of the conjugate pad contacts a
perimeter of the inlet. In some embodiments, the first surface of
the conjugate pad contacts a perimeter of a collar or an
O-ring.
[0162] The device can also comprise an attachment member. In some
embodiments, the attachment member is flexible or made from a
flexible material. In some embodiments, the attachment member is
fixed or made from a non-flexible material. The fixed attachment
member can be, for example, a hinge and the like that can, for
example, contact the conjugate pad or another layer or membrane of
the system and can mediate its displacement. The fixed attachment
member, such as, but not limited to, a fixed hinge or other
compressible material that acts like a hinge and can return to a
shape or dimension upon compression release. The attachment member
can be capable of displacing the conjugate pad. The attachment
member can also just be plastic and although can flex, its flexing
properties are not used in the functioning of the device.
[0163] The flexible material can be, for example, an elastic or
elastomer material. An attachment member can be, for example,
attached to a conjugate pad and/or a hydrophobic membrane. The
attachment member can also be attached to any membrane or member of
the device. Examples of attachment members include, but are not
limited to, elastomer band, rubber band, spring, and the like. In
some embodiments, the attachment member can be made of a shape
memory material. In some embodiments, the attachment member makes
it possible to create a delay between moving the locking member and
moving the conjugate pad or any other type of membrane or pad that
the attachment member is attached to. In some embodiments, the
movement of the pad or membrane does not happen at the same time as
the sliding button or locking member is moved. In some embodiments
of a device that can be used to detect multiple analytes with a
single signal, and not being bound to any particular theory, as the
sliding button or locking member is moved energy is accumulated in
the attachment member and this energy is used to pull on a pad or
membrane that it is attached to the attachment member after the
pressure has been released. In some embodiments, the locking member
is moved away from the force member (i.e., the force member no
longer contacts the locking member) before the conjugate pad is
moved or removed. The conjugate pad, in some embodiments, is moved
once the compression or pressure being exerted by the force member
is completely removed.
[0164] The attachment member can also be attached to either a
sliding button or locking member. The attachment member can be
attached through any means such as, adhesives, staples, tying, and
the like to the other components. In some embodiments, the membrane
or pad has notches in the membrane or pad that allow the attachment
member to attach to the membrane or pad. A non-limiting example can
be seen in FIG. 9.
[0165] In some embodiments of a device that can be used to detect
multiple analytes with a single signal, the housing comprises a
locking member. The locking member can be a slidable locking member
that can move within the device. The locking member can be used to
lock the force member in a position such that the force created by
the force member upon the different layers is maintained. The
locking member is, for example, locking the force member in place
so that the pressure cannot be relieved unless the locking member
is moved to allow the force member to change positions (i.e.
lowered). The locking member, can for example, fit under the head
of the force member, which would keep the force member in the
raised position. The locking member can also be situated so that it
keeps the force member in a particular position (e.g. raised or
lowered). The locking member can be made of any material including,
but not limited to, plastic and the like. The locking member can,
for example, contact the force member either directly or indirectly
through another component that prevents the force member from
releasing the pressure. In some embodiments, the locking member
contacts the force member to compress the conjugate pad.
[0166] The locking member can also contact the attachment member
such that movement of the locking member will move the attachment
member, any other membrane (e.g. conjugate pad, hydrophobic
membrane, test membrane, or absorbent member) or other component
that is attached to the attachment member. For example, if the
locking member is moved to relieve the pressure of the force member
thereby allowing the force member to change positions (e.g. from
raised to a lower position), the movement of the locking member
will also deform/accumulate energy into the attachment member so it
can move the membrane or pad once the pressure has been
sufficiently reduced. When the conjugate pad is attached to the
attachment member and the locking member is moved this will also
move the conjugate pad once the pressure has been sufficiently
reduced. In some embodiments, the pressure is completely removed.
The conjugate pad can be, for example, moved such that it is
removed from the device. In some embodiments, the conjugate pad is
moved to reveal the test membrane through the inlet opening. The
amount of the test membrane that is revealed will depend upon the
type of detection that is used. For a visual detection more of the
test membrane may need to be revealed in the inlet opening. For a
non-visual, e.g. fluorescent, near-infrared, infrared, radioactive
or chemiluminescent detection, less or none of the test membrane
may need to be revealed. In some embodiments, the conjugate pad is
moved so that it no longer can be seen or detected through the
inlet opening. In some embodiments, the movement of the conjugate
pad can create another opening other than the inlet opening to
visualize or detect the test membrane. In some embodiments, the
conjugate pad is dissolved to visualize or detect the test membrane
(e.g. detection of the analyte or multiple analytes with a single
signal). The conjugate pad can be made of a dissolvable material
such that when the conjugate pad comes into contact with the sample
or another solution the conjugate pad partially or completely
dissolves.
[0167] In some embodiments of a device that can be used to detect
multiple analytes with a single signal, the attachment member is
also attached to the impermeable or hydrophobic membrane. When the
attachment member is moved the movement will also move or remove
the impermeable or hydrophobic membrane. As discussed herein, the
presence of the impermeable or hydrophobic membrane can allow the
test sample to dwell or rest upon the test membrane by slowing or
stopping the vertical flow. When the impermeable or hydrophobic
membrane is moved or removed, either by its attachment to the
attachment member or through other means, the vertical flow is no
longer impeded or inhibited.
[0168] In some embodiments of a device that can be used to detect
multiple analytes with a single signal, the housing comprises a
sliding button. A sliding button can also be referred to as a
sliding member. The sliding button can cross the inner and outer
surfaces of the housing. In some embodiments, the sliding button or
sliding member protrudes to an outer surface of the housing. In
some embodiments, the sliding button is attached either directly or
indirectly to the locking member. When the sliding button is
attached (directly or indirectly) to the locking member the
movement of the sliding button also moves the locking member. The
attachment member in some embodiments can be attached to the
sliding button. In some embodiments, the attachment member is
attached to both the sliding button and the locking member. The
sliding button and the locking member can also be constructed as a
single unit.
[0169] In some embodiments of a device that can be used to detect
multiple analytes with a single signal, any one or more of the
inlets comprise an opening chosen from a range of about 0.2 to
about 20 cm.sup.2. In some embodiments, any one or more of the
inlets is about 1 to about 2 cm in diameter. In some embodiments,
any one or more of the inlets is about 1 or about 1.5 cm in
diameter. In some embodiments, any one or more of the inlets is
about 1, about 2, about 3, about 4, or about 5 cm in diameter. In
some embodiments, where there is more than one inlet, the inlets
can be different sizes or the same sizes. The size of each inlet is
independent of one another. In some embodiments of the devices and
systems described herein, the devices or systems comprises 1, 2, 3,
4, or 5 inlets. In some embodiments of the devices and systems
described herein, the devices or systems comprises at least 1, 2,
3, 4, or 5 inlets.
[0170] In some embodiments of a device that can be used to detect
multiple analytes with a single signal, the inlet opening comprise
an opening chosen from a range of about 0.2-20 cm.sup.2. In some
embodiments, the inlet opening is about 1 to about 2 cm in
diameter. In some embodiments, the inlet opening is about 1 or
about 1.5 cm in diameter. In some embodiments, the inlet opening is
about 1, about 2, about 3, about 4, or about 5 cm in diameter.
[0171] In some embodiments of a device that can be used to detect
multiple analytes with a single signal, a device for detecting an
analytes comprises a first member and a second member. In some
embodiments, the first member and second member are in contact with
each other. In some embodiments, the first member comprises one or
more inlets. In some embodiments, between the first and second
member is an analyte detection membrane system. In some
embodiments, the analyte detection membrane system between the
first and second member comprises a conjugate pad, an adhesive
member, a test membrane and an absorbent member. In some
embodiments, the analyte detection membrane system comprises in the
following order: a conjugate pad; an adhesive member; a test
membrane; and an absorbent member. As discussed herein, in some
embodiments, at least a portion of each of the conjugate pad, test
membrane, and absorbent member are substantially parallel to each
other. In some embodiments, at least a portion of each of the
conjugate pad, test membrane, and absorbent member are in a
different spatial plane.
[0172] In some embodiments of a device that can be used to detect
multiple analytes with a single signal, the analyte detection
membrane system is compressed between the first and second member
(e.g. of the force member). In some embodiments, the analyte
detection membrane system is compressed between a plane formed by
the first member and a plane formed by the second member wherein
the planes formed by the first and second members are substantially
parallel to each other and the analyte detection membrane system.
In some embodiments, the planes are parallel to each other and the
analyte detection membrane system. In some embodiments, the first
and second members that compress the analyte detection membrane
system is a force member. For example, the force member can be
referred to as comprising a first and second member to create the
force that compresses the analyte detection membrane system.
[0173] In some embodiments of a device that can be used to detect
multiple analytes with a single signal, the first and second member
are attached to one another along an edge of the first member that
is parallel to an edge of the second member. In some embodiments,
the first and second member are attached by a spring, hinge, and
the like. The manner by which the first and second member are
attached is not limited and can be by any structure that enables
the analyte membrane system to be compressed between the first and
second member. In some embodiments, the first and second member are
contiguous with one another and form a clip. Examples of clips
(e.g. force members) are shown throughout the present application
(e.g. FIG. 16). The clip, can be for example cut from metal or
other type of material that allows the first member to be flexible
such that the analyte detection membrane system can be inserted
between the first and second members. In some embodiments, the
first member is removable.
[0174] In some embodiments of a device that can be used to detect
multiple analytes with a single signal, the first member is
attached or in contact with the conjugate pad, wherein the movement
or removal of the first member moves the conjugate pad or removes
the conjugate pad from the device. In some embodiments, the
conjugate pad is removable.
[0175] In some embodiments of a device that can be used to detect
multiple analytes with a single signal, the conjugate pad is
removed from the device comprising the first and second member by
removing only the conjugate pad.
[0176] In some embodiments of a device that can be used to detect
multiple analytes with a single signal, the conjugate pad comprises
a tab. The tab can be used to remove or to facilitate the removal
of the conjugate pad.
[0177] In some embodiments of a device that can be used to detect
multiple analytes with a single signal, the devices described
herein are placed in a container. In some embodiments, the
container is a pouch or a bag. In some embodiments, the container
comprises an inlet. In some embodiments, the container comprises a
removable or movable member or layer that when moved or removed
exposes the inlet allowing the sample to be applied to the analyte
detection membrane system. Examples of a removable or movable
member or layer includes, but is not limited to, a flap or tab. A
flap or tab, for example, is shown in FIGS. 18 and 19. In some
embodiments, the removable layer or movable layer can also act as a
seal for the container. The seal can protect the conjugate pad
and/or the analyte detection membrane system.
[0178] In some embodiments of the devices and systems described
herein, the removable or movable layer is in contact with or
attached to the conjugate pad.
[0179] In some embodiments of a device that can be used to detect
multiple analytes with a single signal, a device for detecting an
analyte comprises a first outer member and a second outer member
comprising a first inner member and a second inner member, wherein
the first inner member and second inner member are in contact with
each other. In some embodiments, the first outer member comprises
an inlet. In some embodiments, the first inner member comprises an
inlet. In some embodiments, the first outer member and the first
inner member comprise an inlet. In some embodiments, between the
first and second inner members is an analyte detection membrane
system. In some embodiments, the device comprises a conjugate pad.
In some embodiments, the device lacks a conjugate pad. In some
embodiments, the analyte detection membrane system comprises a test
membrane and an absorbent member and optionally a conjugate pad. In
some embodiments, the analyte detection membrane system comprises
in the following order a test membrane and an absorbent member. In
some embodiments, at least a portion of each of the optional
conjugate pad, test membrane, and absorbent member are
substantially parallel to each other. In some embodiments, as
discussed above, the analyte detection membrane system is
compressed between the first inner member and second inner member.
In some embodiments, the device and/or system comprises an adhesive
member as described herein. In some embodiments, the device
comprises a filtration membrane. In some embodiments, the
filtration membrane can be within the analyte detection membrane
system. In some embodiments, the a first surface of the filtration
membrane contacts a surface of the first inner member and a second
surface of the filtration membrane contacts another membrane or
member of the analyte detection membrane system. In some
embodiments, a second surface of a filtration membrane contacts a
surface of a test membrane. The filtration membrane can be any
material as described herein. For example, the filtration membrane,
in some embodiments, can be the same materials that can be a
conjugate pad, test, membrane, absorbent member, and the like. In
some embodiments, the filtration membrane is a glass fiber pad.
[0180] In some embodiments of a device that can be used to detect
multiple analytes with a single signal, where the conjugate pad is
not present within the device or the system, the conjugate is
supplied as a liquid or as a material that can be dissolved in a
liquid (e.g. water, buffered solution, saline, and the like). The
conjugate can be supplied in a separate container (e.g. tube) and
be provided with a device or system described herein. Where the
conjugate is supplied in a container the conjugate is incubated
with the sample before the sample is applied to the analyte
detection membrane system. The sample can be produced by any method
and/or as described herein. For example, a piece of meat can be
swabbed or wiped and to produce a test sample. The test sample can
then be incubated or contacted with the conjugate to produce a test
sample-conjugate mixture. This mixture can then be applied to the
analyte detection membrane system as described herein using a
device and/or system as described herein. In some embodiments, the
test sample-conjugate mixture is applied directly to the test
membrane. In some embodiments, the test sample-conjugate mixture is
filtered or passes through another membrane prior to contacting the
test membrane.
[0181] In some embodiments of a device that can be used to detect
multiple analytes with a single signal, the analyte detection
membrane system is compressed between the first and second inner
members. In some embodiments, the analyte detection membrane system
is compressed between a plane formed by the first inner member and
a plane formed by the second inner member wherein the planes formed
by the first inner member and the second inner member are
substantially parallel to each other and the analyte detection
membrane system. In some embodiments, the planes are parallel to
each other and the analyte detection membrane system. In some
embodiments, the planes are substantially parallel to the first and
second outer members.
[0182] In some embodiments of the devices described herein and
throughout, the conjugate pad is not compressed by the first and
second inner members or by the force members described herein.
[0183] In some embodiments of a device that can be used to detect
multiple analytes with a single signal, the first outer member
comprises a removable or movable tab. In some embodiments, the
conjugate pad is attached to said first outer member. In some
embodiments, the conjugate pad is attached to the removable or
movable tab. In some embodiments, the first outer member and second
outer member form a container and the container encapsulates the
first and inner second member. In some embodiments, the container
is a pouch, bag (e.g. sealable (e.g. zipper, adhesive, and the
like) or any other type of container that can encompass the analyte
detection membrane system and that is compressed between the first
and second inner members.
[0184] In some embodiments of a device that can be used to detect
multiple analytes with a single signal, the container comprises a
removable or movable tab. The removable or movable tab can be any
shape and can be completely removable or removed to an extent that
exposes the inlet. In some embodiments, the tab when moved or
removed removes or moves the conjugate pad. The conjugate pad can
be moved, for example, a sufficient distance so that the results of
the test membrane can be analyzed (e.g. visualized).
[0185] In some embodiments of a device that can be used to detect
multiple analytes with a single signal, a first surface of the
conjugate pad is in contact with the first outer member and a
second surface of the conjugate pad is in contact with the first
inner member.
[0186] In some embodiments of a device that can be used to detect
multiple analytes with a single signal, the first and second inner
members are attached to one another along an edge of the first
inner member that is parallel to an edge of the second inner
member. In some embodiments, the first and second inner members are
attached by a spring, hinge, and the like. The manner by which the
first and second inner members are attached is not limited and can
be by any structure that enables the analyte membrane system to be
compressed between the first and second member. In some
embodiments, the first and second inner members are contiguous with
one another and form, for example, a clip. Examples of clips are
shown throughout the present application. The clip, can be for
example, cut from metal or other type of material that allows the
first inner member to be flexible such that the analyte detection
membrane system can be inserted between the first and second
members. In some embodiments, the first inner member is
removable.
[0187] As discussed herein, the devices and systems can comprise a
removable or movable layer (e.g. tab). The removable or movable
layer can be removed or moved by manual force, such as, but not
limited to, pealing or tearing. The removable or movable layer can
also be removed or moved by mechanical force. The manner by which
the removable or movable layer is moved can by any means. Examples
of a removable or movable layer includes but is not limited to,
tabs, flaps, and the like. As discussed herein, this flap or tab
can act as a seal and the like.
[0188] As discussed herein, the conjugate pad can comprise an
analyte specific capture reagent. In some embodiments, the
conjugate pad comprises a plurality of analyte specific capture
reagents. In some embodiments, the conjugate pad comprises 1, 2, 3,
4, or 5 analyte specific capture reagents. The analyte can be any
molecule that can be specifically recognized by a capture reagent.
Examples of analytes include a polynucleotide molecule (e.g. DNA,
RNA, siRNA, antisense oligonucleotide) a peptide, a protein, a
saccharide, a polysaccharide, a carbohydrate, and the like. The
antigen can also refer to different epitopes present on the same
protein or polypeptide. The analyte can also refer to antigens from
pathogenic or non-pathogenic organisms.
[0189] In some embodiments of a device that can be used to detect
multiple analytes with a single signal, the devices may be housed
singly, in pairs, or in multiple configurations. The housing can be
watertight to prevent leakage and can be manufactured from a
variety of inert materials, such as polymer materials. The inlet,
in some embodiments, can be of sufficient volume to contain any
required amount of sample or reagents to be used with the
invention.
[0190] Because the membranes, members, or pads of the device are,
in some embodiments, chemically inert, they may have to be
activated at any reaction site where it is desired to immobilize a
specific binding reagent against solvent transport. Various methods
may be required to render the reagent immobilized according to the
particular chemical nature of the reagent. Generally, when the
media is nitrocellulose or a mixed nitrocellulose ester, no special
chemical linkage is required for the immobilization of reagents.
Various techniques may be used for other materials and reagents
which include functionalization with materials such as
carbonyldiimidazole, glutaraldehyde or succinic acid, or treatment
with materials such as cyanogen bromide. Other suitable reactions
include treatment with Schiff bases and borohydride for reduction
of aldehyde, carbonyl and amino groups. DNA, RNA and certain
antigens may be immobilized against solvent transport by baking
onto the chromatographic material. Baking may be carried out at
temperatures ranging from about 60.degree. C. to about 120.degree.
C. for times varying from about five minutes to about 12 hours, and
in some embodiments, at about 80.degree. C. for about two
hours.
[0191] Embodiments described herein also provide systems comprising
the devices described herein and a buffer container. The systems
can be used to detect multiple analytes with a single signal. The
buffer container can be any buffer that the sample that is being
tested can be mixed with and then applied to the device. For
example, the sample can be taken from a source and the sample can
be mixed with the buffer. The buffer can be a lysis buffer that
will lyse the cells or a buffer that maintains the pH of the sample
so that the analysis can be done properly. The buffer container can
be any shape and can be included outside or inside the housing of
the device.
[0192] In some embodiments, a system is provided that comprises a
sample collector. The sample collector can be any material that can
take a sample from a source and allow the sample to be tested. For
example, the sample collector can be a swab, such as a cotton-swab.
In some embodiments, the sample collector is an inoculator. In some
embodiments, the housing comprises the sample collector and a
portion of the sample collector is in the inside of the housing. In
some embodiments, the sample collector is partially outside and
partially inside the housing. In some embodiments, the sample
collector is completely outside the housing.
[0193] Kits for detecting multiple analytes with a single signal is
also provided, wherein the kits comprise a device described herein.
The kit can include a device as described herein, a sample
collector, a buffer container, an instruction manual, a positive
control, a negative control, or any combination thereof. With
respect to the kit, a positive control is a sample that is known to
contain the analyte(s) that can be detected with the device present
in the kit. In contrast the negative control, would not contain an
analyte that can be detected by the kit. For example, the negative
control when used in conjunction with the anti-antibody would be
able to demonstrate that the device is working properly.
[0194] Buffers can also be included in the present invention.
Examples of buffers include, but are not limited to, 1.times. PBS
(10 mM Phosphate, 137 mM Sodium Chloride, 2.7 mM Potassium
Chloride), a wash buffer (e.g. 10 mM Sodium Phosphate, 150 mM NaCl,
0.5% Tween-20, 0.05% Sodium Azide), a membrane buffer (e.g. 10mM
Sodium Phosphate, 0.1% Sucrose, 0.1% BSA, 0.2%, PVP-40 pH 7.21,
filtered with 0.2 .mu.m filter.), Polyclonal Conjugate Block Buffer
(e.g. 50 mM Borate, 10% BSA, pH 8.93); Polyclonal Conjugate Diluent
(e.g. 50 mM Borate, 1% BSA, pH 9.09), or Blocking Buffers (e.g. 10
mM Sodium Phosphate, 0.1% Sucrose, 0.025% Silwet pH 7.42; 10 mM
Sodium Phosphate, 1% Sucrose, 1% Trehalose, 0.01% BSA, 0.025%
Tween-20; 0.05% Sodium Azide, 0.025% Silwet pH 7.4; 10 mM Sodium
Phosphate, 0.1% Sucrose, 0.1% BSA, 0.2% PVP-40 pH 7.21). The buffer
can also be, but is not limited to, a blocking buffer (e.g. 10% BSA
in deionized water, pH 7.4 or 1% BSA in deionized water, pH 7.4);
10 mM Borate, 3% BSA, 1% PVP40, and 0.25% Tween-100; and the
like.
[0195] The conjugate pad and the test membrane can be contacted
with any of the buffers described herein either in the presence or
absence of a capture reagent and, in some embodiments, allowed to
dry.
[0196] Examples of buffers that are lysis buffers include, for
example, but are not limited to, 2% Tween (v/v) and 0.1%
Triton(v/v); 2% Tween(v/v) and 0.1% SDS(w/v); 2% Tween(v/v) and
0.1% BSA(w/v); 2% Tween(v/v) and 1% BSA(w/v), 0.1% SDS(w/v), 1%
BSA(w/v), or any combination thereof . The lysis buffers can also
be, for example, 5% Tween/PBS; 2% Tween/PBS +0.1% SDS; 2% Tween/PBS
+1% BSA. Other examples of lysis buffers include, but are not
limited to, 5% Tween-80(v/v); 5% Triton X-100(v/v); 5% NP40(v/v);
2% Tween-80(v/v); 2% Triton X-100(v/v); 2% NP40(v/v); 1%
Tween-80(v/v); 1% Triton X-100(v/v); and 1% NP40(v/v). The
detergents and other components of the buffers can be made with any
suitable buffer suitable for proteins, and includes, but is not
limited to, water and phosphate buffered saline. The lysis buffers
can be used to prepare the samples prior to the samples making
contact with the devices described herein. In some embodiments, a
lysis buffer is not used. A lysis buffer is not used on a sample
when a surface protein or surface analyte is desired to be detected
in the method. Accordingly, in some embodiments, the sample is not
subject to lysis or conditions that would cause a cell to be lysed.
Where a cell is being used the cell could be part of the bridging
complex and replace an amplicon that is shown, for example, in FIG.
3. The cell could be labeled or unlabeled so long as there is a
capture reagent that can create a similar bridging complex.
[0197] The present subject matter also provides for methods of
detecting multiple analytes comprising contacting a sample with a
device and/or system as described herein, wherein the sample
contacts the conjugate pad and the test membrane, wherein a
positive reaction with the test membrane indicates the presence of
the multiple analytes. In some embodiments, the conjugate pad
comprises a signal detection unit or a capture regent that binds to
the signal detection unit. In some embodiments, the test membrane
comprises a second analyte-specific capture reagent. This can bind
to an interaction unit present on the analyte. The sample can have,
for example, the differentially labeled amplicons. For example, the
test membrane can comprise a first capture reagent that binds to an
interaction unit present on the first analyte. The conjugate pad
can have a capture reagent that binds to the signal detection unit.
The analytes can be incubated with the bridging unit and/or the
signal detection unit prior to being applied to the device and
contacting the conjugate pad and/or test membrane. A positive
reaction is indicated when the complex of the analytes, capture
reagents, and signal detection units are present. Otherwise the
signal is not generated. A capture reagent can be applied to the
test membrane so that it will indicate a positive reaction when it
binds to its specific binding partner. The system and devices can
be utilized to form the complexes described herein. For example,
after a PCR reaction occurs that creates differentially labeled
amplification products, the products are incubated with the
antibodies that can be used to create the bridging complex. The
incubation mixture is then added to the device. The sample flows
through the conjugate pad that contains a capture reagent and then
interacts with the test membrane that contains another capture
reagent. In some embodiments, the conjugate pad is removed or moved
so that the signal can be detected. Movement of the conjugate pad
in a vertical flow device is described herein. If all of the
analytes are present, the bridging complex is created and the
single signal can be detected. The specific capture reagent on the
test membrane can be applied in any manner such that when it is
detected it can form a line, a circle, a plus sign, a broken line,
an "X" or any other pattern. In some embodiments, the control line
indicating that the device is working properly will cross the
analyte specific line and when the multiple analytes are present
and detected the detectable label will form a plus sign. The
detection can be determined by the detection of the detection
reagent as described herein and by routine methods known to one of
skill in the art. Similar methods can be used, for example, in an
ELISA system.
[0198] In some embodiments, a sample contacts the device, which is
then followed by a buffer being applied to the device after the
sample has contacted the device. For example, a sample comprising
the analytes can be contacted with the conjugate pad such that the
sample is transferred to the conjugate pad. Following the contact
with the conjugate pad a separate solution can be applied to the
device to facilitate or initiate the vertical flow through the
devices described herein.
[0199] In some embodiments, as described herein, the capture
reagent is an antibody. In some embodiments, the sample that is
tested is a solution but can also be a mixture of solution or
buffer and solid material that can be applied to the device. The
solution will then solubilize the analyte(s) and allow the
conjugate pad's capture reagent to come into contact with the
appropriate analyte present in the sample. In some embodiments, the
sample comprises a cell lysate. In some embodiments, the cell
lysate has been clarified by centrifugation or other means to
remove non-soluble materials.
[0200] In some embodiments, the methods comprise contacting a test
sample with a sample collector and contacting the sample collector
with the device. The test sample can be a sample comprising
amplicons which are created from multiple analytes. In some
embodiments, the methods comprise contacting the sample collector
with a solution or buffer, wherein the solution or buffer is
applied to the device. In some embodiments, the samples are
contacted with the conjugate pad prior to the sample coming into
contact with the test membrane. In some embodiments, the sample is
contacted with the conjugate pad and the test membrane
simultaneously.
[0201] In some embodiments, the method comprises moving the
conjugate pad of the devices described herein, wherein the movement
of the devices exposes the test membrane for detection. In some
embodiments, the locking member moves the conjugate pad. In some
embodiments, the conjugate pad is attached to the locking member
and/or the sliding button member. In some embodiments, movement or
removal of the removable member moves or removes the conjugate pad.
In some embodiments, the conjugate pad is attached to the removable
member and/or the adhesive member. In some embodiments, when the
removable member is moved or removed the adhesive member is also
moved with respect to its original position or removed from the
device. The analyte can be those that are discussed herein or any
other analyte that can be detected using the methods and devices
described herein. In some embodiments, the method comprises
applying the sample to the device and allowing the sample to flow
through the device via vertical flow.
[0202] In some embodiments the detection or indication of the
presence or absence of multiple analytes occurs in less than 60
seconds. In some embodiments, the detection or indication of the
presence or absence of multiple analytes occurs in about 30 to
about 60 seconds. In some embodiments, the detection or indication
of the presence or absence of multiple analytes occurs in less than
2 minutes. In some embodiments, the detection or indication of the
presence or absence of multiple analytes occurs in about 30
seconds.
[0203] In some embodiments, devices for detecting multiple analytes
with a single signal are provided. In some embodiments, the device
comprises a housing. The device can comprise a first housing member
and a second housing member to form the housing. In some
embodiments, the first and second housing members are separate
members. The first and second housing members can be manufactured
as a single piece. The single piece, in some embodiments, can be
separated into the two housing members to allow for the
introduction of the materials into the housing (e.g. device). In
some embodiments, the device comprises an inlet. The inlet can be
in either housing member (e.g. first or second housing member). The
inlet can be oriented above the conjugate pad, such that a sample
that is introduced into the device through the inlet contacts the
conjugate pad prior to contacting the test membrane. The device is
oriented such that regardless of any pressure being applied to the
device, the sample will flow vertically down through the layers of
membranes (e.g. analyte detection membrane system). Accordingly, in
some embodiments, the second housing member comprises the inlet
opening. In some embodiments, the second housing member is on top
of the first housing member. The inlet can be any size or shape as
described herein so long as the size and shape is sufficient for
the introduction of a sample into the device such that the sample
can contact the analyte detection membrane system.
[0204] The device can comprise one or more force members. The force
members can apply pressure to the analyte detection membrane
system. The force is applied perpendicular or substantially
perpendicular to the membranes or layers of the analyte detection
membrane system. In some embodiments, the device comprises at least
1, 2, 3, 4, or 5 force members. In some embodiments, the device
comprises at least 1, 2, 3, 4, or 5 force members. In some
embodiments, the device comprises a plurality of force members. The
force members can be in contact with a housing member. In some
embodiments, a first surface of the force member is in contact with
a housing member (e.g. first or second housing member). In some
embodiments, a second surface of the force member contacts the
analyte detection membrane system. As described herein, the force
member can be used to compress the analyte detection membrane
system to facilitate the flow of the sample through the analyte
detection membrane system. The pressure can facilitate the sample
to flow vertically through the analyte detection membrane system.
The force members can be oriented in the device independently of
one another. The force members can also be manipulated such that
each force member applies a pressure to a distinct analyte
detection membrane system and that the force applied to each
analyte detection membrane system is different or, in some
embodiments, the same or substantially the same.
[0205] In some embodiments of a device that can be used to detect
multiple analytes with a single signal, the device comprises one or
more movable locking members. In some embodiments, the movable
locking member contacts a force member. In some embodiments, the
movable locking member contacts each force member present in the
device. For example, in a device comprising a first and second
force members, the movable locking member is in contact with the
first force member and the second force member. The movable locking
member, in some embodiments, supports the force member such that
the force member is in a raised position. The raised position can
be determined by comparing the force member's position when it is
in contact with the movable locking member to when the force member
is not in contact with the movable locking member. In the absence
of contact between the force member and the movable locking member,
the force member is in a first position. When the movable locking
member is in contact with the force member, the force member is in
a second position. In some embodiments, the second position of the
force member is considered to be a raised position. The raised
position can be used to compress the layers (e.g. membranes) of the
analyte detection membrane system. When the movable locking member
is not in contact with the force member, in some embodiments, the
analyte detection membrane system is not compressed.
[0206] The device can comprise one or more movable locking members.
In some embodiments, the device comprises a plurality of, or 1, 2,
3, 4, or 5 movable locking members. In some embodiments, the device
comprises at least 1, 2, 3, 4, or 5 movable locking members. In
some embodiments, the device comprises a number of movable locking
members that is equal to the number of force members present in the
device.
[0207] The movable locking members can also comprise a moving
member, such as, but not limited to, a handle. The moving member
can be used, for example, to turn or move the movable locking
member such that the locking member contacts the force member. In
some embodiments, the moving member disengages the locking members
from the force member such that the force member changes positions
(e.g. from a raised position to a lower position). The moving
member can be used to relieve or apply the pressure being applied
on the analyte detection membrane system. The moving member can
also be used to relieve or apply compression of the analyte
detection membrane system. In some embodiments, the moving member
rotates the locking member around a central axis of the device. For
example, after applying the sample to the device and the sample
flows through at least one analyte detection membrane system, the
moving member is moved, which rotates the movable locking member in
either a clockwise or counterclockwise direction. The rotation of
the movable locking member allows the force member to be lowered
into a different position. The rotation of the movable locking
member can also allow the pressure that is applied to the analyte
detection membrane system to be relieved. In some embodiments, the
pressure is completely relieved, or, in some embodiments, the
pressure is only partially relieved.
[0208] In some embodiments of a device that can be used to detect
multiple analytes with a single signal, the moving member that
moves the movable locking member protrudes through the first or
second housing member. In some embodiments, the moving member is
accessible through the moving member outlet. In some embodiments,
the moving member rotates around a central axis of the device when
moved. In some embodiments, the moving member moves the movable
locking member laterally (e.g. horizontally) or vertically. In some
embodiments, the movable locking member moves laterally (e.g.
horizontally) or vertically when moved.
[0209] The moving member and the movable locking member can be
constructed as a single piece or as two pieces. In some
embodiments, where the movable locking member and the moving member
are two separate pieces and are constructed to interact with one
another such that the movement of one moves the other. For example,
one of the two pieces can have a "male member" that protrudes from
the surface and inserts into the "female member" of the other piece
to form the interaction.
[0210] The movement of the movable locking member by the moving
member can also be used to move or remove the conjugate pad present
in the analyte detection membrane system. As discussed herein, the
conjugate pad can be removed to allow visualization or the analysis
of the test membrane. The conjugate pad, as discussed herein, can
be removed completely from the analyte detection membrane system or
an amount that is sufficient to allow visualization or analysis of
the test membrane. Analysis of the test membrane can be based
solely upon visual inspection, or in some embodiments, an optical
reader can be used to analyze the test membrane to determine the
absence or presence of an antigen in the sample.
[0211] In some embodiments of a device that can be used to detect
multiple analytes with a single signal, the device comprises a
plurality, or two or more analyte detection membrane systems. In
some embodiments, the device comprises at least 1, 2, 3, 4, or 5
analyte detection membrane systems. In some embodiments, the device
comprises 1, 2, 3, 4, or 5 analyte detection membrane systems. The
analyte detection membrane system can be as described herein and
throughout the present application.
[0212] In some embodiments of a device that can be used to detect
multiple analytes with a single signal, the device comprises one or
more flexible or non-flexible attachment members. In some
embodiments, the device comprises a plurality of flexible or
non-flexible attachment members. In some embodiments, the device
comprises at least 1, 2, 3, 4, or 5 flexible or non-flexible
attachment members. In some embodiments, the device comprises 1, 2,
3, 4, or 5 flexible or non-flexible attachment members. In some
embodiments, the flexible or non-flexible attachment member contact
the movable locking member. In some embodiments, the flexible or
non-flexible attachment member contact the movable locking member
and the conjugate pad. The flexible or non-flexible attachment
member can be used to remove or move the conjugate pad away from
the rest of the layers (e.g. membranes) of the analyte detection
membrane system. In some embodiments, the device comprises a number
of flexible or non-flexible attachment members that is equal to the
number of analyte detection membrane systems present in the device.
In some embodiments, the device comprises a number of flexible
attachment members that is equal to the number of force members
present in the device. The flexible or non-flexible attachment
members can also be used to retract the conjugate pad so as to
reveal or expose a portion or all of the test membrane.
[0213] For example, in some embodiments, a device comprises three
analyte detection membrane systems and three force members. A
device with more than one analyte detection membrane system can be
used to detect different analytes or different multiple analyte
sets. In such a device, for example, the device comprises a first,
second, and third attachment member. The first attachment member
can be in contact with the conjugate pad of the first analyte
detection membrane system and a movable locking member.
Additionally, in some embodiments, the second attachment member can
be in contact with the conjugate pad of the second analyte
detection membrane system and a movable locking member. In some
embodiments, the third attachment member can be in contact with the
conjugate pad of the third analyte detection membrane system and a
movable locking member. In some embodiments, the first, second, and
third attachment members are in contact with the same movable
locking member. In some embodiments, the first, second, and third
attachment members are in contact with different movable locking
members. For example, in some embodiments, the first and second
attachment members are in contact with the same movable locking
member and the third attachment member is in contact with a
different movable locking member. Each attachment member is
independently contacted with one or more movable locking
members.
[0214] In some embodiments of a device that can be used to detect
multiple analytes with a single signal, the movable locking member
comprises one or more movable locking member extensions. In some
embodiments, the movable locking member extensions contacts a force
member. In some embodiments, the device comprises a number of
movable locking member extension that is the same as the number of
force members that are present in the device. In some embodiments,
the movable locking member extension partially encircles or
encompasses the force member. In some embodiments, the movable
locking member extension completely encircles or encompasses the
force member. The shape of the movable locking member or member
extension can be any shape to keep the force member in a raised
position. In some embodiments, the extension is a hook or hook-like
shape that partially or completely encircles or encompasses the
force member. The shape is not essential so long as the shape acts
as a support for the force actuator (e.g. force member).
[0215] The number of movable locking member extensions can the same
or different as the number of force members present in a device
described herein. In some embodiments, a device comprises a
plurality of movable locking member extensions. In some
embodiments, a device comprises at least 1, 2, 3, 4 or 5 movable
locking member extensions. In some embodiments, a device comprises
1, 2, 3, 4 or 5 movable locking member extensions. For example, in
some embodiments, a device comprises a first, second, and third
force members attachment members and a first, second, and third
movable locking member extensions. In this non-limiting example,
for example, the first force member contacts the first movable
locking member extension, the second force member contacts the
second movable locking member extension, and the third force member
contacts the third movable locking member extension.
[0216] In some embodiments of a device that can be used to detect
multiple analytes with a single signal, the movable locking member
comprises an attachment member extension, which can be flexible or
inflexible. In some embodiments, the attachment member extension
contacts the attachment member. In some embodiments, the attachment
member extension comprises an attachment member extension nodule.
The nodule can be any shape or size that allows the attachment
member to be secured to so that the attachment member securely
maintains its contact with the movable locking member. In some
embodiments, the one or more movable locking member extensions
extend radially (e.g. outward) from the center of the movable
locking member.
[0217] The number of attachment member extension can the same or
different as the number of analyte detection membrane systems
present in a device described herein. In some embodiments, a device
comprises a plurality of flexible or non-flexible attachment member
extensions. In some embodiments, a device comprises at least 1, 2,
3, 4 or 5 flexible or non-flexible attachment member extensions. In
some embodiments, a device comprises 1, 2, 3, 4 or 5 flexible or
non-flexible attachment member extensions. For example, in some
embodiments, a device comprises a first, second, and third
attachment members and a first, second, and third attachment member
extensions. In this non-limiting example, for example, the first
attachment member contacts the first attachment member extension,
the second attachment member contacts the second attachment member
extension, and the third attachment member contacts the third
attachment member extension.
[0218] In some embodiments, the devices described herein comprise
flexible and non-flexible attachment members and/or member
extensions. Throughout the present disclosure, reference made be
made to an attachment member or member extensions that are flexible
or non-flexible. If one embodiment discloses a flexible member it
is understood that another embodiment is also disclosed where the
member is non-flexible unless context dictates to the contrary.
[0219] In some embodiments of a device that can be used to detect
multiple analytes with a single signal, the device comprises a
channel system. The channel system can be used to transport the
sample (e.g. fluid) from the inlet opening of the device to the
analyte detection membrane system(s) present in the device. As used
herein, the "channel system" refers to the entire system regardless
of how many individual channels are a part of the system. For
example, the channel system can comprises two or more channels,
such as, but not limited to, capillaries, that transport fluid from
the inlet to an analyte detection membrane system. In some
embodiments, the channel system comprises one or more branches
(e.g. arms). The one or more branches can be transport fluid to one
or more analyte detection membrane systems. In some embodiments,
the channel system comprises 1, 2, 3, 4, or 5 branches. In some
embodiments, the device comprises a number of branches in the
channel system that is equal to the number of analyte detection
membrane systems present in the device.
[0220] In some embodiments of a device that can be used to detect
multiple analytes with a single signal, each branch of the channel
system comprises capillary tubes that transport the fluid from the
inlet to the analyte detection membrane system. In some
embodiments, each branch comprises a plurality of capillary tubes.
In some embodiments, each branch comprises at least 1, 2, 3, 4, or
5 capillary tubes. In some embodiments, the channel system does not
comprise capillary tubes or tube-like formations but is made from a
material that allows a portion of the sample to be transported from
the inlet to the conjugate pad of the analyte detection system. In
some embodiments, the channel system is a porous material that can
be used to transport the sample from the inlet to the analyte
detection membrane system. In some embodiments, the channel system
is made from the same material as the conjugate pad. In some
embodiments, the channel system and the conjugate pad are a
contiguous piece of material. In some embodiments, the channel
system comprises a Porex material. These porous materials allow the
inlet to be in fluid communication with the analyte detection
membrane system. In some embodiments, the porous material comprises
polyethylene, polypropylene, polytetrafluourouethylene (PTFE),
PVDF, ethyl vinyl acetate, Nylon 6, thermoplastic polyurethane
(TPU), SCP, and the like. In some embodiments, the conjugate pad
and the channel system are the same materials or different
materials. In some embodiments, the channel system does not
comprise a porous material and/or a capillary tube system.
[0221] In some embodiments of a device that can be used to detect
multiple analytes with a single signal, the channel system contacts
the inlet. In some embodiments, the channel system contacts the top
of the analyte detection membrane system. In some embodiments, the
channel system contacts the top of the conjugate pad or a membrane
that is on top of the conjugate pad. In some embodiments, the
channel system contacts an edge of the conjugate pad or an edge of
a membrane that is on top of the conjugate pad. Regardless of how
the sample contacts the analyte detection membrane system, in some
embodiments, the sample flows vertically through analyte detection
membrane system. Therefore, although the sample may flow
horizontally (e.g. laterally) from the inlet to the analyte
detection membrane system, the sample is not analyzed until it
flows vertically through the analyte detection membrane system.
This is distinctly different from lateral flow systems where a
sample flows laterally (e.g. horizontally) through multiple
membranes or test layers.
[0222] In some embodiments of a device that can be used to detect
multiple analytes with a single signal, the channel system divides
the sample into equal portions, wherein each equal portion contacts
an independent analyte detection membrane system. In some
embodiments, the channel system divides the sample into one or more
unequal portions. The one or more unequal portions are then
transported to independent analyte detection membrane systems.
[0223] For example, in a device that comprises a first and second
analyte detection membrane systems the device comprises a channel
system that comprises a first and second branch. In some
embodiments, the first branch contacts the first analyte detection
membrane system and the second branch contacts the second analyte
detection membrane system. Upon application of the sample to the
device (e.g. through the inlet opening), the sample is transported
in equal portions through the first and second branches of the
channel system to the first and second analyte detection membrane
systems. In some embodiments, the sample is transported in unequal
portions through the first and second branches of the channel
system to the first and second analyte detection membrane systems.
The sample can be divided into unequal portions, for example, based
upon the number of capillaries present in each branch. For example,
the first branch can comprise more capillaries than the second
branch. The greater number of capillaries will allow more of the
sample to be transported through the first branch than the second
branch, thereby delivering unequal portions to the first and second
analyte detection membrane systems.
[0224] Accordingly, the branches of the channel system may have the
same number of capillaries or different numbers of capillaries. The
numbers of capillaries in each branch of the channel system is
independent of each branch. That is each branch of the channel
system can have the same number or a different number of
capillaries as another branch. Therefore, in some embodiments, the
device's channel system can be described as a capillary channel
system. In some embodiments, the channel system is enclosed in a
channel housing that is separate and distinct from the first and
second housing members described herein for the device itself. In
some embodiments, the channel housing is transparent, translucent,
opaque, or partially translucent.
[0225] As discussed herein, the test membrane can be analyzed
either visually with the human eye or through a machine, such as an
optical reader to determine the presence or absence of multiple
analytes with a single signal. In some embodiments, the analysis is
done through a portal. In some embodiments, the device comprises
one or more portals that are sufficient in size to allow
visualization of a test membrane of one or more of the analyte
detection membrane systems. In some embodiments, a single portal is
used to visualize each of the test membranes present in the device.
In some embodiments, the device does not comprise a portal. In
embodiments, where the device does not comprise a portal, the test
membrane can still be visualized by using a transparent or
translucent housing for the device. In some embodiments, the first
and/or second housing are transparent or translucent. Where the
first and/or second housings are transparent or translucent this
can allow an analyte detection membrane systems and its test
membrane when it is revealed upon moving or removing the conjugate
pad. In some embodiments, the device comprises a plurality of
portals. In some embodiments, the device comprises at least 1, 2,
3, 4, or 5 portals. In some embodiments, the device comprises 1, 2,
3, 4, or 5 portals. In some embodiments, a device comprises 1
portal that is continuous and exposes each analyte detection
membrane system present in the device to visual inspection.
[0226] As discussed herein, the force members can be allowed to
move between at least two positions (e.g. raised or lowered;
engaged or disengaged). In some embodiments, the force member is
lowered and is encompassed by a force actuator outlet. Thus, in
some embodiments, the device comprises one or more force actuator
outlets that that can accept the force member as it is lowered. In
some embodiments, the device comprises a plurality of force
actuator outlets. In some embodiments, the force actuator outlet is
a groove. In some embodiments, the force actuator outlet is a
circle or substantially circular. The force actuator outlet can be
used to suspend the force actuator (e.g. force member) at a
particular position. The force actuator outlet can also be used to
retain the force actuator in a second position. In some
embodiments, the circumference of the force actuator outlet is
greater than the circumference of the portion of the force member
that is entering the outlet. In some embodiments, the circumference
of the force actuator outlet is greater than the largest
circumference of the force member. In some embodiments, the
circumference of the force actuator outlet is not greater than the
largest circumference of the force member, wherein the force member
has areas with at least two different circumferences. For example,
force members are described herein that would have two different
circumferences. A force member can comprise a cap with one
circumference and a support structure that supports the cap with a
different circumference. The support structure can, in some
embodiments, have a smaller circumference than the cap. In some
embodiments, the force actuator outlet can have a circumference
that is larger than the support structure circumference, but
smaller than the cap structure circumference. In some embodiments,
the number of force actuator outlets is the same or different than
the number of the force members present in a device.
[0227] The force actuator outlet can also be a continuous
depression in a housing member that can accept each and every force
member in the device when it is lowered and no longer compressing
the analyte detection membrane system. The outlet can be used to
temporarily house the force member or it can be permanent, such
that the force member cannot be raised again to compress or further
compress the analyte detection membrane system.
[0228] As discussed herein and throughout the present application,
the conjugate pad, permeable membrane, test membrane, and absorbent
member can be or are compressed by the force member under certain
forces as described herein and including, but not limited to a
force from about 1 lbf to about 100 lbf. In some embodiments, where
there are multiple analyte detection membrane systems, the pressure
applied to each membrane detection system can be different or it
can be the same. For example, in a device that has a first, second,
and third analyte detection membrane system, the first analyte
detection membrane system can be compressed under a force of 5 lbf,
the second analyte detection membrane system can be compressed
under a force of 10 lbf, and the third analyte detection membrane
system can be compressed under a force of 25 lbf. In another
example, in some embodiments, the first and second analyte
detection membrane systems are compressed under the same pressure
and the third analyte detection membrane system is compressed under
a pressure that is different from the first and second analyte
detection membrane systems. The differences in pressure can be used
to use different flow rates, which can be useful for different
analytes. The pressure is correlated with the flow rate. The
pressure can be manipulated by the position of the force member and
how much the layers of the analyte detection membrane system are
compressed. The specific force used can be determined and measured
by one of skill in the art using known and routine methods.
[0229] As described herein, in some embodiments, the present
invention provides a system comprising any device described herein,
a buffer container and/or a sample collector. In some embodiments,
the present invention provides a kit comprising any device
described herein and one or more of a positive control, a negative
control, an instruction booklet, a buffer container, and/or a
sample collector, or any combination thereof.
[0230] The methods described herein can be used with a device that
has, for example, a plurality, two or more, analyte detection
membrane systems. The methods can be also be used with devices that
have 2, 3, 4, or 5 analyte detection membrane systems. Where there
are more than two analyte detection membrane systems (e.g. 3, 4, 5,
6, 7, 8, 9, or 10) the methods and the descriptions contained
herein are modified to be consistent with the number of analyte
detection membrane systems. These changes are made in accordance
with the descriptions contained herein and any routine changes that
would be known by one of skill in the art. The changes to encompass
more than 2 analyte membrane detections systems based upon the
descriptions contained herein combined with knowledge of one of
skill in the art would not require undue experimentation. In some
embodiments as described herein, the device comprises two or more
analyte detection membrane systems. In some embodiments, the method
comprises contacting a sample (e.g. the sample comprising multiple
analytes) with the device and a portion of the sample being
transported through a channel system to the conjugate pads of the
two or more analyte detection membrane systems. In some
embodiments, the method comprises detecting a positive or negative
reaction for the analytes, wherein a positive reaction indicates
that the presence of the multiple analytes. In some embodiments,
the two or more analyte detection membrane systems are compressed
by the force member. In some embodiments, the sample vertically
flows from the conjugate pad to the test membrane. In some
embodiments, the method further comprises compressing the analyte
detection membrane system by the force member. In some embodiments,
the method comprises moving the conjugate pad of the two or more
detection systems after a portion of the sample has contacted and
flowed through the conjugate pad, thereby exposing the test
membrane for analysis. In some embodiments, the test membrane is
exposed within the portal opening for detection. In some
embodiments, the conjugate pad of the two or more detection systems
is moved by moving the movable locking member. In some embodiments,
the moving the movable locking member comprises rotating the
movable locking member around the central axis of the device. In
some embodiments, the movable locking member is moved laterally or
vertically. In some embodiments, the moving lockable member moves
the conjugate pad of the two or more detection systems
simultaneously or sequentially. In some embodiments, the method
further comprises relieving the compression of the two or more
analyte detection systems. The pressure can be relieved or
lessened, for example, by moving the movable locking member. In
some embodiments, the movable locking member is moved (e.g.
rotated) by turning or moving the moving member that is connected
to the movable locking member.
[0231] In some embodiments, one or more of the analyte detection
membrane systems are compressed prior to the sample contacting the
channel system. In some embodiments, one or more of the analyte
detection membrane systems are compressed prior to the sample
coming into contact with the conjugate pad of the one or more of
the analyte detection membrane systems. In some embodiments, each
of the analyte detection membrane systems is compressed
simultaneously. In some embodiments, each of the analyte detection
membrane systems is compressed independently. In some embodiments,
each of the analyte detection membrane systems present in a device
is compressed prior to a sample coming into contact with the
conjugate pad.
[0232] In some embodiments, the method comprises relieving the
pressure applied by a force member on the two or more analyte
detection membrane systems, wherein said force member moves
vertically or horizontally to relieve said pressure. In some
embodiments, the method comprises the force member moving from a
first position to a second position to relieve the pressure. In
some embodiments, the force member moves into or comes into contact
with a force actuator outlet when the movement of the force member
relieves or reduces the pressure or relieves or reduces the force
being applied to the analyte detection membrane system. In some
embodiments, the force member drops partially or completely out of
the device.
[0233] In some embodiments of a device that can be used to detect
multiple analytes with a single signal, the present invention
provides a device for detecting an analyte comprising a pressure
actuator, a pressure release, an analyte detection membrane system,
an analyte detection membrane system receptacle, and an outlet. In
some embodiments, the analyte detection membrane system receptacle
is of sufficient size and shape to accept the analyte detection
membrane system. In some embodiments, the receptacle is a groove.
In some embodiments, the receptacle is a case that can be, but not
necessarily, removed from the device.
[0234] In some embodiments, the analyte detection membrane system,
as described herein, can be encompassed or contained within a
cartridge or housing. The housing can comprise a first and/or
second housing member. In some embodiments, where the analyte
detection membrane system is contained within a housing or a
cartridge, the receptacle is of sufficient size and shape to accept
the housing or the cartridge. In some embodiments, the housing or
cartridge comprises an inlet. The inlet can be used to apply the
sample to the analyte detection membrane system. In some
embodiments, the cartridge or housing comprises a second outlet
that allows the sample to flow through and out of the housing and
cartridge. The analyte detection membrane system can be any analyte
detection membrane system as described herein.
[0235] In some embodiments of a device that can be used to detect
multiple analytes with a single signal, the device comprises a
pressure actuator. The pressure actuator, for example, can be the
force member that is described in herein. In some embodiments, the
pressure actuator is an air valve or vacuum valve that either
applies air pressure to the analyte detection membrane system or
draws a vacuum through the analyte detection membrane system. In
some embodiments, the pressure actuator can be regulated by a
pressure release or pressure regulator. The pressure release or
pressure regulator can be, for example, a vacuum release. The
release or regulator can be used to regulate the pressure or vacuum
being applied to the analyte detection membrane system. The
pressure or vacuum can be applied to the analyte detection membrane
system through an outlet or tube that is present in the device. The
outlet can be the same outlet present in the cartridge or housing
described herein or it can be a different outlet or tube. The
outlet or tube can be used so that the pressure or vacuum to be
applied with specificity rather than allowing it to diffuse across
the entire device.
[0236] In some embodiments, the housing (e.g. cartridge) encasing
the analyte membrane detection comprises an upper housing and a
lower housing. In some embodiments, the housing comprises a
plurality of membrane or pad holders. In some embodiments, the
housing comprises one or more membrane or pad holders. In some
embodiments, the housing comprises 1, 2, 3, 4, or 5 membrane or pad
holders. In some embodiments, the housing comprises at least 1, 2,
3, 4, or 5 membrane or pad holders. In some embodiments, the
housing comprises an inlet. In some embodiments, the housing
comprises an outlet. In some embodiments, the vacuum actuator
directly or indirectly contacts the housing outlet.
[0237] In some embodiments of a device that can be used to detect
multiple analytes with a single signal, the device and any device
described herein comprises an ejector for ejection the housing. The
ejector can be used to facilitate the removal of the housing that
contains the analyte detection membrane system. In some
embodiments, the devices comprise a housing separator. The housing
separator can be used to facilitate the separation of the housing.
In some embodiments, the ejector can also act as the housing
separator.
[0238] In addition to the methods described herein, in some
embodiments, a method of detecting multiple analytes comprises
applying a sample containing the multiple analytes to a device
comprising a pressure actuator, a pressure regulator, an analyte
detection membrane system, an analyte detection membrane system
receptacle, and an outlet or any other device or analyte detection
membrane system described herein. In some embodiments, the sample
is contacted with the analyte detection membrane system, wherein
the sample vertically flows through the analyte detection membrane
system. In some embodiments, the method comprises detecting the
presence or absence of the analyte. This can be done according to
the bridging complex that is formed through the use of the
interaction units, capture reagents, and signal detection units
described herein.
[0239] In some embodiments of using the devices to detect multiple
analytes, detecting comprises removing or moving the conjugate pad
present in the analyte detection membrane system a sufficient
amount to visualize the result, wherein a positive result indicates
the presence of the multiple analytes. In some embodiments,
detection comprises removing the analyte detection membrane system
from the device and further removing or moving the conjugate pad a
sufficient amount to visualize the detection of the analyte or
multiple analytes with a single signal. In some embodiments, the
analyte detection membrane system is contained within a housing or
cartridge, and therefore, in some embodiments, the housing or
cartridge is removed from the device prior to the movement or
removal of the conjugate pad. In some embodiments, the housing is
separated into a first (e.g. upper) and a second (e.g. lower)
housing prior to the removal or movement of the conjugate pad as
described herein. In some embodiments, the separation of the
housing into a first and a second housing removes or moves the
conjugate pad to visualize the test membrane as described herein.
In some embodiments, the housing is separated manually and/or
mechanically. In some embodiments, the housing (e.g. cartridge) is
ejected from the device. In some embodiments, the housing is
ejected from the device by an ejector. In some embodiments, the
housing is separated by a separator. In some embodiments, the
ejector also functions as a separator.
[0240] In some embodiments, the method comprises applying pressure
on or drawing a vacuum through an analyte detection membrane
system. In some embodiments, the method comprises releasing or
reducing the pressure or the vacuum. In some embodiments, the
pressure or vacuum is released or reduced by using the pressure
regulator. In some embodiments of the methods described herein, the
sample that is contacted with the analyte detection membrane system
flows through the analyte membrane system at a flow rate that is
regulated by a pressure actuator. In some embodiments, the entire
sample flows through the analyte detection membrane system at a
constant rate. In some embodiments, the sample flows through the
analyte detection membrane system at a variable rate. In some
embodiments, the variable rate comprises at least one period of
time where the flow rate of a portion of the sample is 0. For
example, the pressure being applied or vacuum being drawn can be
regulated such that the sample stops flowing through the analyte
detection membrane system for a period of time. This can be
referred to as a "dwell." As described elsewhere in the present
document, the dwell can be implemented by placing impermeable or
slightly impermeable membranes between the conjugate pad and the
other layers of the analyte detection membrane system. The dwell,
however, can also be regulated by regulating (e.g. changing) the
pressure that is applied to the analyte detection membrane system.
The dwell can also be regulated by regulating (e.g. changing) the
vacuum that is being drawn through the analyte detection membrane
system. Any method of regulating the flow rate through the analyte
detection membrane system, including but not limited to, the flow
rate through the conjugate pad and/or the test membrane can be
used.
[0241] The devices herein, can also be automated or used in
conjunction with an optical reader or other type of spectrometer.
The advantages of combining the systems and devices described
herein with an optical reader or other type of spectrometer is that
the sensitivity of the devices and assays can be increased such
that less analyte present in the sample is necessary to identify a
sample as being positive for that analyte. This greater sensitivity
can be then be used, for example, to determine if food contains
pathogens, a person has a certain disease or condition, or if a
product has an analyte that is otherwise undetectable using other
devices and methods in the same amount of time it takes to use the
presently described methods and devices.
[0242] Accordingly, in some embodiments of a device that can be
used to detect multiple analytes with a single signal, the present
invention provides a device for detecting multiple analytes
comprising a sample inlet, an analyte detection cartridge
receptacle, an analyte detection cartridge receptacle inlet, an
optional conjugate pad remover, a pressure actuator, a spectrometer
(e.g. optical reader), a display unit, a signal processing unit.
The pressure actuator can be a force member whose position is
modified to regulate the pressure being applied to the analyte
detection membrane system that is used in conjunction with a
device. The pressure actuator can also regulate the pressure by
drawing a vacuum through the analyte detection membrane system that
is used in conjunction with a device. The spectrometer can be any
spectrometer that can detect the presence of a signal. The signal
can be an optical signal. The signal can be a signal that is
emitted in a spectrum chosen from, for example, infrared spectrum;
near-infrared spectrum; visible spectrum, x-ray spectrum,
ultra-violet spectrum, gamma rays, electromagnetic spectrum, and
the like.
[0243] The spectrometer can be connected to the signal processing
unit (e.g. computer). The signal processing unit can take the
signal that is transmitted to it and analyze the signal to
determine the presence or absence of the sample. An example of a
signal processing unit is, but not limited to, a computer. The
signal processing unit can programmed to analyze the signal
transmitted by the spectrometer. The programming can implement an
algorithm to analyze the signal to determine the presence or
absence of an analyte in the sample. The algorithm can be based
upon criteria that are pre-installed in the signal processing
unit's memory or that are entered by the user of the device. The
types of information that can be entered can be cut-offs for a
positive or negative signal, processing times, and the like. The
signal processing unit can also be used to regulate the pressure
applied to or the vacuum drawn through the analyte detection
membrane system.
[0244] The signal processing unit can be used or programmed to
regulate the flow rate of the sample through the analyte detection
membrane system. The flow rate can be regulated by regulating the
pressure that is applied to or a vacuum that is drawn through the
analyte detection membrane system. As described above with respect
to the methods described herein, the sample that is contacted with
the analyte detection membrane system flows through the analyte
membrane system at a flow rate that is regulated by a pressure
actuator. The pressure regulator can be in turn regulated by, for
example, the signal processing unit. In some embodiments, the
entire sample flows through the analyte detection membrane system
at a constant rate, which is regulated by the signal processing
unit. In some embodiments, the sample flows through the analyte
detection membrane system at a variable rate, which is regulated by
the signal processing unit. In some embodiments, the variable rate
comprises at least one period of time where the flow rate of a
portion of the sample is 0, which can be regulated by the signal
processing unit. For example, the pressure being applied or vacuum
being drawn can be regulated by the signal processing unit such
that the sample stops flowing through the analyte detection
membrane system for a period of time. As discussed herein, this can
be referred to as a "dwell." The dwell, for example, can be
regulated by regulating (e.g. changing) the pressure that is
applied to the analyte detection membrane system, which can be
implemented or controlled by the signal processing unit. The dwell
can also be regulated by regulating (e.g. changing) the vacuum that
is being drawn through the analyte detection membrane system, which
can be implemented or controlled by the signal processing unit. Any
method of regulating the flow rate through the analyte detection
membrane system, including but not limited to, the flow rate
through the conjugate pad and/or the test membrane can be used and
such method can be regulated or implemented by the signal
processing unit.
[0245] In some embodiments of a device that can be used to detect
multiple analytes with a single signal, the devices described
herein and throughout, comprises an analyte detection cartridge
receptacle positioning member. The detection cartridge receptacle
positioning member can be used, for example, to place the analyte
detection membrane system in the proper position to accept the
sample and/or for the sample to be analyzed. In some embodiments,
the system is positioned for spectrometer analysis. The detection
cartridge receptacle positioning member can be, in some
embodiments, motorized and/or controlled by the signal processing
unit. In some embodiments, the detection cartridge receptacle
positioning member is not motorized but can controlled by a manual
controller, such as, but not limited to a lever or screw that
allows that receptacle's position to be modified. In some
embodiments, the signal processing unit controls the movement of
the analyte membrane detection receptacle by moving the analyte
membrane detection receptacle moving member. In some embodiments,
the analyte detection cartridge receptacle positioning member is in
contact with analyte detection cartridge receptacle.
[0246] In some embodiments of a device that can be used to detect
multiple analytes with a single signal, the devices described
herein can comprise a waste receptacle. The waste receptacle can be
in the interior of the device or outside but still contacting the
device. The waste receptacle can accept analyte detection membrane
systems that have been used. In some embodiments, as described
herein, the analyte detection membrane system is contained in a
housing (e.g. cartridge). The housing can then be ejected into the
waste receptacle. The ejection can be manual or automated. In some
embodiments, the ejection is controlled by a signal processing
unit. In some embodiments, the signal processing unit controls an
ejector that ejects the analyte detection membrane system from the
analyte detection membrane system receptacle into the waste
receptacle. Like all of the devices described herein, in some
embodiments, the device comprises an analyte detection membrane
system, which can or cannot be encased in a housing (e.g.
cartridge).
[0247] In some embodiments of a device that can be used to detect
multiple analytes with a single signal, the pressure actuator
contacts the analyte detection membrane system. In some
embodiments, the pressure actuator is attached to the device at a
point that allows movement of the pressure actuator. In some
embodiments, the pressure actuator is attached at a pivot point
that allows the pressure actuator to pivot at a single contact
point.
[0248] In some embodiments, the devices described herein comprise a
display. In some embodiments, the display is an electronic display.
In some embodiments, the signal processing unit receives an input
from the spectrometer and displays information on the display unit.
The display unit can be display various information, including but
not limited to, the presence and/or absence of one or more
analytes, status, and the like.
[0249] In some embodiments, the present invention comprises
detecting the multiple analytes using a device comprising a signal
processing unit or a device described herein. In some embodiments,
the method comprises contacting the device with a sample that
contacts the analyte detection membrane system within the device.
The sample then flows through the analyte detection membrane
system. In some embodiments, the method comprises detecting the
presence or absence of the analyte. In some embodiments, the
detecting comprises the optical reader detecting an optical signal
from the analyte membrane system, the optical reader communicating
the optical signal to the signal processing unit, the signal
processing unit analyzing the optical signal to determine the
presence or absence of the analyte; and the signal processing unit
displaying a result on the display unit. The displayed result can
be visual and/or audible. The signal analyzed can be a signal in a
spectrum chosen from infrared spectrum; near infrared spectrum;
visible spectrum, x-ray spectrum, ultra-violet spectrum, gamma
rays, or electromagnetic spectrum. In some embodiments, the signal
is in the near-infrared spectrum. In some embodiments, the method
comprises ejecting the analyte detection membrane system into a
waste receptacle. In some embodiments, the signal processing unit
is a computer.
[0250] Referring to the drawings, in some embodiments, FIGS. 1
through 36 depict embodiments of devices, components of such
representative devices, and various views of such embodied devices
that can be used in the methods and/or in conjunction with or
without other devices and/or systems described herein.
[0251] These devices described herein are non-limiting and any
other device, including other vertical flow devices, can be used to
detect multiple analytes according to the methods described herein
using the bridging complexes that are created using the various
labels and capture reagents.
[0252] FIG. 8 depicts a device that can be used to detect multiple
analytes with a single signal comprising a first housing member
(10), a buffer container (15), a second housing member (20), a
groove for the sliding button (25), a sliding button (30), an inlet
opening (35), a collar (40), and a test membrane (45). FIG. 8
depicts a test membrane (45) comprising two capture reagents. The
first (10) and second (20) housing members can also be referred to
as the lower and upper housing members, respectively. In FIG. 1,
the sample would be applied through the inlet opening (35) and can
be allowed to vertically flow through to the test membrane (45). In
FIG. 8, the groove (25) allows the sliding button to move, which
when attached to the locking member moves the locking member and
can, in some embodiments, move the conjugate pad and change the
position of the force member.
[0253] FIG. 9 depicts a device that can be used to detect multiple
analytes with a single signal comprising a first housing member
(10), a second housing member (20), a groove for the sliding button
(25), a sliding button (30), an inlet opening (35), a collar (40),
a test membrane (45), a conjugate pad (50), a plurality of
absorbent members (e.g. pads) (55), an attachment member (60), a
locking member (65), and a force member (70). FIG. 9 depicts the
conjugate pad (50), test membrane (45) and absorbent pad (55)
arranged substantially parallel to one another. The force member
(70) when in contact with the absorbent member would be applying
pressure that is substantially perpendicular to the conjugate pad.
As can be seen in FIG. 9, a sample that is contacted with the
device through the inlet opening (35) would flow vertically through
the conjugate pad (50) to the test membrane (45). Not explicitly
shown in FIG. 9, but in some embodiments, a the permeable membrane
is also substantially parallel to the conjugate pad (50) and to the
test membrane (45), with a first surface of the permeable membrane
contacting a surface of the conjugate pad (50) a second surface of
the permeable membrane contacting a surface of the test membrane
(45).
[0254] FIG. 10 depicts a conjugate pad (50), a permeable membrane
(75), a test membrane (45), and a plurality of absorbent members
(55) that can be used to detect multiple analytes with a single
signal. FIG. 10 depicts the components that can be used to detect
multiple analytes with a single signal being substantially parallel
with one another. FIG. 10 depicts the permeable membrane (75)
comprising an opening. This opening can be used to allow
visualization and detection of the test membrane's results.
[0255] FIG. 11 depicts a device that can be used to detect multiple
analytes with a single signal comprising a first housing member
(10), a buffer container (15), a second housing member (20), a
sliding button (30), a test membrane (45), a conjugate pad (50), a
permeable membrane (75), a plurality of absorbent members (e.g.
pads) (55), an attachment member (60), a locking member (65), and a
force member (70). FIG. 11 also depicts the force member (70)
comprising a shaft (72) and a head (71) where the head (71) is
wider than the shaft (72).
[0256] FIG. 12 depicts a partial view of a device that can be used
to detect multiple analytes with a single signal comprising a first
housing member (10), a locking member (65), a sliding button (30),
and force member (70). FIG. 12 depicts the locking member (65) in
contact with the force member (70) such that the force member (70)
is in a raised method. FIG. 12 also depicts the movement of the
locking member (65) and the sliding button (30) away from the force
member (70) allowing the force member to change positions. In some
embodiments, the change in position is that the force member is
lowered.
[0257] FIG. 13 depicts a side cut away view of a device that can be
used to detect multiple analytes with a single signal comprising a
first housing member (10), a second housing member (20), a sliding
button (30), a locking member (65), a collar (40), an O-ring (41),
a force member (70), and a support for the force member (73). The
support for the shaft can be, for example, part of the first
housing member (10) and is shaded differently for example purposes
only. FIG. 13 depicts the button (30) in contact with the locking
member (65) in such a way that movement of the button (30) will
move the locking member (65). Movement of the locking member (65)
will take away the support from the force member (70), which would
allow the force member (70) to change positions. FIG. 13 also
depicts the shaft (72) and the head (71) of the force member. The
head (71) creates a lip where the locking member (65) can slide
under and support the force member (70).
[0258] FIG. 14 depicts a partial view of a device that can be used
to detect multiple analytes with a single signal comprising a first
housing member (10), a second housing member (20), an inlet opening
(35), a test membrane (45), a conjugate pad (50), a plurality of
absorbent members (55), an attachment member (60), a locking member
(65), and a force member (70). FIG. 8 depicts the attachment member
(60) attached to the conjugate pad (50) and the locking member
(65). FIG. 14 also depicts the conjugate pad being compressed
against the second housing member (20) and the perimeter of the
inlet opening (35). FIG. 14 depicts the head of the force member
(71) applying the pressure by contacting the plurality of absorbent
members (55). In FIG. 14, a sample can be applied to the device
through the inlet opening (35) so that the sample contacts the
conjugate pad (50) and because of the pressure the sample through
vertical flow contacts the test membrane (45).
[0259] FIG. 15A depicts a partial view of a device that can be used
to detect multiple analytes with a single signal comprising a first
housing member (10), a second housing member (20), an inlet opening
(35), a test membrane (45), a conjugate pad (50), a plurality of
absorbent members (55), an attachment member (60), a locking member
(65), and a force member (70). FIG. 8 depicts the movement of the
locking member (65), which is attached to the attachment member
(60). The movement of the attachment member (60), which is attached
to the conjugate pad (50) moves the conjugate pad. FIG. 15A depicts
the test force member (70) changing positions and a lessening or
elimination of the pressure and/or compression of the test membrane
(45). FIG. 15C and 15D also depicts the movement of the conjugate
pad (50) away from the inlet opening (35) revealing the test
membrane (45) for visualization and/or detection.
[0260] FIG. 16 depicts an attachment member (60) attached to a
conjugate pad (50). FIG. 16 depicts notches (51) in the conjugate
pad (50) as locations for the attachment member (60) to attach to.
The attachment member can also be attached through other means such
as through adhesives, staples, and other forms of attachment.
[0261] FIG. 17 depicts a partial view of device that can be used to
detect multiple analytes with a single signal comprising a second
housing member (20), a plurality of pads or membranes (80), wherein
the plurality of pads comprises a test membrane, a permeable
membrane, and one or more absorbent members, and retaining members
(85) that can retain the plurality of pads or membranes (80). FIG.
10 depicts the structures that when the conjugate pad is moved the
plurality of pads remains in place. Any means or other structure
can be used to keep the plurality of pads in place.
[0262] FIG. 18 depicts a representative device that can be used to
detect multiple analytes with a single signal comprising a first
housing member (1002) that further comprises a housing inlet
(1006), and a second housing member (1004). In some embodiments,
the first and second housing members can be constructed as a single
unit. The housing inlet allows for the introduction of a sample
onto the components inside the housing. The housing inlet can be of
sufficient size to handle an appropriate amount of volume of a
solution that is added to the device. In some embodiments, the size
of the opening created by the housing inlet is sufficient to handle
about 0.1 to about 3 ml, about 0.1 to about 2.5 ml, about 0.5 to
about 2.0 ml, about 0.1 to about 1.0 ml, about 0.5 to about 1.5 ml,
about 0.5 to about 1.0 ml, and about 1.0 to about 2.0 ml. In some
embodiments, the dimensions of the device are such that any
dimension (e.g., width, depth, or height) is less than or equal to
about 5.08 cm (2.000 inches). In some embodiments, the height of
the device is less than about 0.635 cm (0.250 inches), less than
about 0.254 cm (0.100 inches), less than about 0.191 cm (0.075
inches), less than about 0.165 cm (0.065 inches), less than about
0.152 cm (0.06 inches), or less than about 0.140 cm (0.055 inches).
In some embodiments, the height of the device is about 0.127 cm
(0.050 inches). In some embodiments, the width or depth of the
device is less than or equal to about 5.08 cm (2.000 inches), about
4.83 cm (1.900 inches), about 4.699 cm (1.850 inches), about 4.572
cm (1.800 inches), about 4.445 cm (1.750 inches), about 4.191 cm
(1.650 inches), about 4.064 cm (1.600 inches), or about 3.81 cm
(1.500 inches). In some embodiments, the device is about 0.127 cm
(0.050 inches) in height, about 4.445 cm (1.750 inches) in depth,
and about 3.81 cm (1.500 inches) in width.
[0263] In some embodiments, the device that can be used to detect
multiple analytes with a single signal comprises a plurality of
components comprising one or more of: a removable member, a
conjugate pad, an adhesive member, a test membrane, an absorbent
member, a force member, a support member, or any combination
thereof
[0264] In some embodiments, the device that can be used to detect
multiple analytes with a single signal comprises a force member, a
removable member, a conjugate pad, a test membrane, an adhesive
member and/or an absorbent member. In some embodiments, the device
comprises an analyte detection membrane system. In some
embodiments, the analyte detection membrane system comprises a
conjugate pad, a test membrane, and an absorbent member. In some
embodiments, the analyte detection membrane system comprises an
additional permeable membrane, but the device can also be free of a
permeable membrane. In some embodiments, the analyte detection
membrane system comprises in the following order: a conjugate pad,
an adhesive member, a test membrane, and an absorbent member.
[0265] FIG. 19 depicts an exploded view of the inside of a
representative device that can be used to detect multiple analytes
with a single signal comprising a removable member (1005), a
conjugate pad (1050), an adhesive member (1010), a test membrane
(1030), an absorbent member (1040), and a support member (1020),
wherein the support member further comprises an optional support
member inlet (1025). The removable member and the adhesive member
can also comprise optional removable member inlet (1008) and
adhesive member inlet (1012), respectively. Such components could
reside within, for example, the device of FIG. 18.
[0266] FIG. 20 depicts representative components of another
representative device that can be used to detect multiple analytes
with a single signal comprising an adhesive member (1010), a
support member (1020), a test membrane (1030), and an absorbent
member (1040). As can be seen in FIG. 20, a sample can flow through
the adhesive member (1010) and contact the test membrane
(1030).
[0267] FIG. 21 depicts an adhesive member (1010), a support member
(1020), a test membrane (1030), and an absorbent member (1040).
FIG. 21 depicts the components being substantially parallel with
one another. FIG. 21 further depicts the support member (1020)
comprising a support member inlet (1025). This inlet can be used to
allow the sample to vertically flow through the device.
[0268] FIG. 22 depicts, in part, a conjugate pad (1050), a test
membrane (1030), and an absorbent member (1040). FIG. 22 also
depicts the conjugate pad in contact and/or attached to a removable
member (1005). FIG. 22 also depicts the removable member being
removed or moved away from the device that can be used to detect
multiple analytes with a single signal, which also removes or moves
away from the device the conjugate pad. The movement of the
conjugate pad allows the test membrane to be visualized, which
facilitates analysis and detection of analytes, including multiple
analytes with a single signal.
[0269] FIG. 23 depicts examples of force members (e.g. clips).
Representative force members can come in a variety of shapes,
sizes, and configurations, but each member applies pressure on the
components that are placed in or on the force member. Each force
member can also comprise an opening (+) into which the analyze
sample is applied. FIG. 23 depicts non-limiting examples of force
members with a first member (110) and a second member (100).
[0270] FIGS. 24A, 24B, 24C, and 24D depict, in part, a force member
comprising a first member (110), b) a second member (100), an inlet
(115), and an analyte detection membrane system (120). FIGS. 24A
and 24B also depict, in part, a conjugate pad (1050). The conjugate
pad is not seen in FIGS. 24C and 24D. FIGS. 24C and 24D also
depict, in part, a test membrane (1030) that is part of the analyte
detection membrane system. FIG. 24D also depicts in part, a test
membrane (1030) that has been reacted with a control, which is
visualized by the band.
[0271] FIG. 25 depicts, in part, a container comprising a removable
or movable tab (200), an inlet (210), a conjugate pad (1050), and
the tab of the conjugate pad (1050). The tab of the conjugate pad
(255) can be used to remove the conjugate pad (1050) from the
device to expose the test membrane. For example, a user could pull
the tab of the conjugate pad (255) to remove the conjugate pad
(1050) from the container. What is not visualized is the analyte
detection membrane system that is compressed between a first member
(110) and a second member (100) as described herein.
[0272] FIG. 26 depicts, in part, a first outer member (310), a
second outer member (320), a movable or removable tab (330), and a
conjugate pad (1050). The movable or removable tab (330) comprises
an inlet that exposes the conjugate pad (1050) so that the sample
can be applied to the conjugate pad. FIG. 26 does not show the
first inner member (110) and the second inner member (100)
compressing the analyte detection membrane system (120). The
removable or movable tab (330) when moved or removed, moves or
removes the conjugate pad (1050), which allows the test membrane to
visualized and analyzed.
[0273] The removable member inlet within the removable member
allows the introduction of a sample onto the conjugate pad. The
inlet can be of sufficient size to handle an appropriate amount of
volume of a solution that is added to the device. In some
embodiments, the size of the inlet is large enough to handle about
0.1 to about 3 ml, about 0.1 to about 2.5 ml, about 0.5 to about
2.0 ml, about 0.1 to about 1.0 ml, about 0.5 to about 1.5 ml, about
0.5 to about 1.0 ml, and about 1.0 to about 2.0 ml. The removable
member can also be constructed such that a portion of the removable
member is permeable to solutions (i.e., the area defined by the
removable member inlet) and another area is impermeable. The
permeable area can act as an inlet because it would allow solutions
to cross the removable member and contact the conjugate pad. The
removable member inlet can have any one of numerous shapes and
sizes. In some embodiments, the first housing member serves as the
removable member. In other embodiments, the first housing member
and the removable member are separate components. In embodiments
where the first housing member and the removable member are
separate components, at least a portion of the housing inlet and
removable member inlet overlap such that a solution can enter
through both inlets.
[0274] In some embodiments, the removable member contacts a first
surface of a conjugate pad. The removable member can also be
attached to the conjugate pad. The removable member can be attached
to the conjugate pad by any means such that when the removable
member is removed from the device or its position is changed, the
conjugate pad is also removed or the position of the conjugate pad
is also changed. The removable member can be attached to the
conjugate pad with, for example, but not limited to, an adhesive.
Adhesives include, but are not limited to, glue, tape, or other
substance that would allow the removable member and the conjugate
pad to be attached to one another.
[0275] The removable member, in some embodiments, directly contacts
the conjugate pad or indirectly contacts the conjugate pad through
another layer. The sample can be, in some embodiments, directly
applied to the conjugate pad through the opening in the removable
member.
[0276] FIG. 27A depicts, in part, an overhead view of a device that
can be used to detect multiple analytes with a single signal
comprising a plurality of portals (2036), an inlet (2035), and a
housing member (2010). FIG. 27A also depicts, in part, a portion of
the channel system (2300) that is visible through the portal
(2301). FIG. 27B depicts, in part, an enlarged area of the device,
specifically, the portal (2036). In the portal one can also see a
plurality of capillary tubes (2301).
[0277] FIG. 28 depicts an underneath view of a device that can be
used to detect multiple analytes with a single signal comprising a
plurality of force actuator outlets (2200), a housing member
(2020), and a moving member (2100).
[0278] FIG. 29 depicts, in part, a first housing member (2010), a
second housing member (2020) a plurality of portals (2036), an
inlet (2035), a channel system (2300), a plurality of capillary
tubes (2301), a conjugate pad (2050), a plurality of test membranes
(2045), and movable locking member (2065). The channel system
depicted in FIG. 29 is depicted as consisting 3 branches, which is
equal to the number of analyte detection membrane systems present
in the device.
[0279] FIG. 30 depicts, in part, a second housing member (2020), a
channel system (2300), a plurality of capillary tubes (2301), a
conjugate pad (2050), a test membrane (2045), and an absorbent
membrane (2055), and a movable locking member (2065), a flexible
attachment member (2060), an analyte detection membrane system
(2400)
[0280] FIG. 31A depicts, in part, a plurality of force actuator
outlets (2200), a channel system (2300), a plurality of capillary
tubes (2301), a plurality of force members (2070), a movable
locking member (2065), a plurality of movable locking member
extensions (2068), a conjugate pad (2050), a plurality of flexible
or non-flexible attachment member extensions (2066) and nodule
(2067), a test membrane (2045), and absorbent membrane (2055).
[0281] FIG. 31B depicts, in part, a similar portion of the device
shown in FIG. 24A, however, the movable locking member (2065) has
been rotated around a central axis and the movable locking member
extension (2068) no longer supports the force member (2070) and the
force member has receded or dropped into the force actuator outlet
(2200).
[0282] FIG. 32 depicts, in part, an exploded view of a device that
can be used to detect multiple analytes with a single signal
comprising a channel system (2300), a conjugate pad (2050), a test
membrane (2045), a plurality of force members (2070), a movable
member (2100) that can turn the movable locking member depicted
(2065). FIG. 32 also depicts, in part, movable locking member
extension (2068), a plurality of flexible or non-flexible
attachment member extensions (2066) and nodule (2067), a flexible
attachment member (2060), an outlet (2105), a second housing member
(2020), a plurality of force actuator outlets (2200), and a portion
of an analyte detection membrane system (2047). The area comprising
the portion of the analyte detection membrane system (2047) has
been enlarged and depicts, in part, a force member (2070), a test
membrane (2045), an absorbent member (2055), and portion of the
movable locking member extension (2068).
[0283] FIG. 33 depicts, in part, a housing (2020), a capillary
channel (2301) and the channel system (2300). A portion of FIG. 33
has been enlarged to depict the conjugate pad (2050), the absorbent
member (2055), and a plurality of capillary tubes (2301).
[0284] FIG. 34 depicts, in part, a cross-sectional view of a device
that can be used to detect multiple analytes with a single signal
comprising a plurality of portals (2036), an inlet (2035), a
movable locking member (2065), a movable member that can move the
movable locking member (2100), a force member (2700), a force
actuator outlet (2200), a plurality of absorbent members (2055), a
test membrane (2045), and a movable locking member extension
(2068). FIG. 34 also depicts an exploded view of a portion of the
analyte detection membrane system comprising a conjugate pad
(2050), a permeable membrane (2056), and an absorbent member
(2055).
[0285] FIG. 35 depicts, in part, a non-limiting example of a
movable locking member (2065) and a movable locking member
extension (2068).
[0286] FIG. 36 depicts, in part, an exterior view and an interior
view of a housing comprising a plurality of portals (2036) and an
inlet (2035).
[0287] FIG. 37 depicts, in part, an interior view and an exterior
view of a housing comprising a plurality of force actuator outlets
(2200) and a movable member outlet (2105).
[0288] FIG. 38 depicts, in part, a device comprising a cartridge
(3100) that can encompass an analyte detection membrane system, a
force actuator (3200) and force release (3000), and outlet (3400),
and an analyte detection membrane system receptacle (3300).
[0289] FIG. 39 depicts, in part, an enlarged view of the outlet
(3400), the receptacle (3300), and the cartridge (3100) depicted in
FIG. 31.
[0290] FIG. 40 depicts, in part, an exploded view of a cartridge
(3100) comprising a first housing member (3110), an inlet (3135), a
conjugate pad (3350), a second housing member (3120), and a
plurality of a membrane holders (3122).
[0291] FIG. 41 depicts, in part, a device for detecting an analyte
comprising an inlet (3335), a membrane system receptacle (3300),
and display (3500).
[0292] FIG. 42 depicts, in part, the interior of the device that
can be used to detect multiple analytes with a single signal
depicted in FIG. 41. The device comprises a cartridge comprising an
analyte detection membrane system (3100), a membrane system
receptacle (3300), a force actuator (3200), a spectrometer (e.g.
optical reader or photodetector (3600), an optional conjugate pad
remover (3201), an optional waste receptacle (3606), a motor and
membrane system receptacle mover (3605/3607).
[0293] FIG. 43, shows the interior of a device that can be used to
detect multiple analytes with a single signal depicted in FIGS. 41
and 42 at various stages of use with the same components depicted
in FIG. 35. FIG. 43A depict the cartridge being inserted into the
receptacle. FIG. 43B depicts the receptacle holding the cartridge
being moved beneath the inlet for sample application and FIG. 43C
depicts the sample being analyzed by the spectrometer.
[0294] FIG. 44 depicts an exploded view of a device that can be
used for the detection of a plurality of analytes with a single
signal comprising a first housing member (10), a second housing
member (20), a groove for the sliding button (25), a sliding button
(30), an inlet opening (35), a test membrane (45), a conjugate pad
(50), an additional membrane (51), an adhesive (52), a plurality of
absorbent members (e.g. pads) (55), an attachment member (60), a
locking member (65), and a force member (70). The components can be
assembled as described and/or shown herein to make a device that
can detect analytes using vertical flow.
[0295] FIG. 45 depicts a partially exploded view of a device that
can be used for the detection of a plurality of analytes with a
single signal comprising a first housing member (10), a second
housing member (20), a groove for the sliding button (25), a
sliding button (30), an inlet opening (35), a test membrane not
seen, a conjugate pad (50), a plurality of absorbent members (e.g.
pads) (not shown), an attachment member (60), a locking member (not
shown), and a force member (not shown). Other variations of this
device can also be made and used in accordance with the methods
described herein.
[0296] The embodiments are now described with reference to the
following examples. These examples are provided for the purpose of
illustration only and the embodiments should in no way be construed
as being limited to these examples, but rather should be construed
to encompass any and all variations which become evident as a
result of the teaching provided herein. Those of skill in the art
will readily recognize a variety of non-critical parameters that
could be changed or modified to yield essentially similar
results.
EXAMPLES
Example 1
[0297] Two separate PCR reactions were performed with Shiga toxin
genes as the template that generated amplicons labeled with: 1)
digoxigenin and biotin, and 2) FITC and biotin. The amplicons were
then either mixed together in the presence or absence of
streptavidin (bridge unit) or ran separately in a rapid flow
through assay: Sample A) amplicon 1 alone, Sample B) amplicon 2
alone, or Sample C) amplicon 1+amplicon 2 with and without
streptavidin. The flow through assay consisted of a solid support
(nitrocellulose membrane) coated with anti-digoxigenin (first
capture reagent) and colloidal gold particles coated with anti-FITC
antibody. In this context, only Sample C with streptavidin
generated a single positive test signal whereas Sample A and Sample
B, or Sample C without streptavidin, resulted in a negative
test.
Example 2
Detecting Multiple Analytes Using Amplicon Bridging
Materials
[0298] PCR reagents: OneTaq Hot Start Polymerase (New England
Biolabs); 5.times. Standard Reaction buffer; Haptenated MHALT1.RV
(Integrated DNA Technologies (IDT)); Haptenated MgC.CH1AS (IDT);
INV018.7E4 V.sub.H gene template (ZG); dNTPs; dH.sub.2O.
[0299] PCR was performed in a standard theremocycler at a ramp rate
of 3-4.degree. C./s. PCR reactions were run through a vertical flow
assay such as those described herein including the Veriflow
Cassette (Invisible Sentinel).
[0300] Amplicons were generated according to standard protocols.
One amplicon was generated dual labeled with fluorescein
isothiocyanate (FITC) and tetramethylrhodamine (TAMRA) and another
amplicon was generated that is dual labeled with TAMRA and
digoxigenin (DIG). The DNA amplicons from the PCR reaction can be
optionally precipitated. If precipitation was performed, it was
done by either by EtOH or Isopropanol +1/10 v Sodium Acetate 3M, pH
5.2 precipitation. To facilitate precipitation, 1 uL of tRNA
glycogen can also be added. The precipitation was allowed to take
place at -20.degree. C. for a minimum of 2 hours or -80.degree. C.
for 15 mins. Precipitated DNA was centrifuged at top speed for
about 15 minutes. Supernatant was discarded the DNA pellet was
allowed to air dry 15 mins. An optional second rinse with 20 uL ICE
cold 70% EtOH can be performed followed by centrifugation and
drying. DNA pellet was suspended with TE (Tris-HC1/EDTA) and the
DNA was allowed to rehydrate for about 24 hours at room
temperature. The amplicons generated were generic sequences and not
specific to any particular bacteria.
[0301] The amplicons were mixed with a biotinylated antibody
recognizing FITC and an antibody that recognized rhodamine (i.e.
the TAMRA label). The mixture can be incubated for longer period of
times, e.g. 5, 10, 15, 20, 25, or 30 min, but such longer times
were not necessary. The incubated mixture was added to a Veriflow
Cassette (vertical flow device), which contained a test membrane
comprising an unlabeled anti-digoxigenin antibody and a conjugate
pad containing streptavidin-gold conjugate. The device detected the
presence of the bridged complex, which contains both amplicons with
a single signal (the colloidal gold). The appropriate controls were
performed and the colloidal gold was only detected when all
components necessary to create the bridging complex were present.
Without wishing to be bound to any particular theory FIG. 3
illustrates the complex that can be formed with the different
components. When bridging complex is formed (see, FIG. 3) the
colloidal gold signal is detected. Other types of detectable
signals can also be used. If one of the amplicons is not present no
signal was detected. After the sample is run through the device the
streptavidin-colloidal gold complex is released from the conjugate
pad and the conjugate pad is removed. Examples of how to make and
use the vertical flow devices can be found herein and in U.S. Pat.
Nos. 8,012,770, 8,183,059 and U.S. patent application Ser. No.
13/500,997, Ser. No. 13/360,528, Ser. No. 13/445,233, each of which
is hereby incorporated by reference in its entirety. These results
demonstrate that two analytes can be specifically detected with a
single detectable signal, which in this example was colloidal gold.
The detection of the signal was not dependent upon precipitating
the amplicons after performing the PCR reaction step.
[0302] The disclosures of each and every patent, patent
application, publication, and accession number cited herein are
hereby incorporated herein by reference in their entirety.
[0303] While this invention has been disclosed with reference to
specific embodiments, it is apparent that other embodiments and
variations of this invention may be devised by others skilled in
the art without departing from the true spirit and scope of the
invention. The appended claims are intended to be construed to
include all such embodiments and equivalent variations.
* * * * *