U.S. patent application number 14/337805 was filed with the patent office on 2015-01-15 for molecular diagnostic assay device and method of use.
This patent application is currently assigned to Credo Biomedical Pte Ltd.. The applicant listed for this patent is Credo Biomedical Pte Ltd.. Invention is credited to Stephen Chang-Chi Kao, Winston Wong, JR..
Application Number | 20150017639 14/337805 |
Document ID | / |
Family ID | 47883995 |
Filed Date | 2015-01-15 |
United States Patent
Application |
20150017639 |
Kind Code |
A1 |
Wong, JR.; Winston ; et
al. |
January 15, 2015 |
Molecular Diagnostic Assay Device And Method Of Use
Abstract
Lateral flow devices and methods of use for a molecular
diagnostic assay are provided. The method is suitable for detection
or monitoring of targets, including biological, chemical, and
material targets that exist in very low concentrations in
biological samples. The methods and devices of the present
application are amenable to power source-free point of care
testing.
Inventors: |
Wong, JR.; Winston; (Sentosa
Island, SG) ; Kao; Stephen Chang-Chi; (Sentosa
Island, SG) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Credo Biomedical Pte Ltd. |
Singapore |
|
SG |
|
|
Assignee: |
Credo Biomedical Pte Ltd.
|
Family ID: |
47883995 |
Appl. No.: |
14/337805 |
Filed: |
July 22, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13618694 |
Sep 14, 2012 |
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14337805 |
|
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61535874 |
Sep 16, 2011 |
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Current U.S.
Class: |
435/6.11 ;
435/287.2; 435/7.92 |
Current CPC
Class: |
G01N 33/558 20130101;
G01N 2021/752 20130101; G01N 33/54386 20130101; G01N 2458/15
20130101; G01N 21/80 20130101; G01N 2021/757 20130101; G01N
2333/918 20130101; G01N 33/581 20130101 |
Class at
Publication: |
435/6.11 ;
435/287.2; 435/7.92 |
International
Class: |
G01N 33/58 20060101
G01N033/58 |
Claims
1. A method of detecting an analyte in a test sample, the method
comprising; i) providing a lateral flow assay device that comprises
a chromatographic medium that includes: (a) a sample loading zone
located upstream of a detection zone; (b) a reporting carrier zone
located between the sample loading zone and a detection zone,
wherein said reporting carrier zone comprises a reporting carrier
capable of forming a complex with the analyte said reporting
carrier comprising a carrier and one or more proficient enzyme
cassettes; and (c) a detection zone, wherein the detection zone
comprises a capture component for the analyte and an indicator; ii)
Contacting the sample application zone with the test sample,
wherein the test sample travels through the reporting carrier zone
along the chromatographic medium from the sample loading zone to
the detection zone and beyond the detection zone; iii) adding a
substrate to the detection zone wherein the substrate undergoes a
reaction in the presence of proficient enzyme analyte containing
reporting carrier; and iv) generating a response of the indicator
within the detection zone that corresponds to the presence or
absence of the analyte in the test sample.
2. The method of claim 1, wherein the lateral flow device further
comprises a control zone down stream of the detection zone and the
control zone comprises a capture component for the reporting
carrier and an indicator; adding a substrate to the control zone
wherein the substrate undergoes a reaction in the presence of
proficient enzyme containing reporting carrier; and generating a
response of the indicator within the control zone that corresponds
to the presence or absence of the reporting carrier.
3. The method of claim 1 further comprising maintaining a reaction
temperature.
4. The method of claim 1, wherein the analyte is a protein.
5. The method of claim 1, wherein the analyte is a small
molecule.
6. The method of claim 1, wherein the analyte is a nucleic
acid.
7. The method of claim 1, wherein the proficient enzyme conjugate
comprises an antibody.
8. The method of claim 1, wherein the proficient enzyme conjugate
comprises a nucleic acid.
9. The method of claim 1, wherein the proficient enzyme conjugate
comprises a proficient enzyme selected from the group consisting of
urease, phosphocholine phosphatase, betagalactosidase, xylose
reductase, shikimate dehydrogenase, malate dehydrogenase,
carboxylesterase, neopullulanase, subtilisin, 4-phytase,
acetylcholinesterase, laccase, bacterial leucyl aminopeptidase,
tripeptidyl-peptidase I, coagulation factor Vila, trypsin,
betafructofuranosidase.
10. The method of claim 3, where the reaction temperature is
maintained to about 4.degree. C., to about 95.degree. C., or higher
while still maintaining enzyme activity.
11. The method of claim 7, wherein the antibody interacts
non-covalently with a proficient enzyme to form the proficient
enzyme conjugate.
12. The method of claim 8, wherein the nucleic acid is covalently
attached to a proficient enzyme to form the proficient enzyme
conjugate.
13. The method of claim 7, wherein the antibody is covalently
attached to a proficient enzyme to form the proficient enzyme
conjugate.
14. The method of claim 1, further providing one or more inactive
pro-enzymes in the detection zone.
15. The method of claim 1, wherein the activity of the proficient
enzyme conjugate is detected by monitoring the effect of the
proficient enzyme assisted reaction.
16. The method of claim 15, wherein the effect of the proficient
enzyme assisted reaction is proton release.
17. The method of claim 16, wherein the proton release produces a
pH change.
18. The method of claim 17, wherein the pH change is determined
using a pH sensitive potassium
I-hydroxy-4-[1-(2-hydroxyethylsulphonyl)phenylazo]-naphthalene-2-sulphona-
te indicator polymer that is immobilized on the membrane.
19. The method of claim 17, wherein the pH change is determined
using a pH sensitive cellulose acetate coupled dye.
20. The method of claim 1, wherein the activity of the proficient
enzyme conjugate is detected by colorimetric change.
21. The method of claim 1, wherein the activity of the proficient
enzyme conjugate is detected by fluorescence emission.
22. The method of claim 1, wherein the activity of the proficient
enzyme conjugate is detected by electrochemical methods.
23. The method of claim 18, wherein the colorimetric change is due
to silver ion reduction.
24. The method of claim 1, wherein the activity of the proficient
enzyme conjugate is detected by precipitation of a soluble
component.
25. The method of claim 24, wherein the soluble component is a
protein or pH sensitive polymer.
26. The method of claim 25, wherein the protein is BSA.
27. The method of claim 25, wherein the pH sensitive polymer is
selected from the group consisting of methyl acrylic acid, methyl
methacrylate, methacrylic acid 2-(dimethylamino) ethyl ester, and
N-hydroxymethyl acrylamide.
28. The method of claim 1 further comprising adding a pre-reporter
carrier to the test sample prior to contacting the sample
application zone with the test sample.
29. The method of claim 1 further comprising an absorptive pad at
the distal end of the chromatographic medium.
30. The method of claim 1, wherein the substrate is allyl hexanoate
and proficient enzyme is carboxylesterase.
31. A lateral flow assay device for detecting the presence of an
analyte within a test sample, the lateral flow assay device
comprising: a chromatographic medium that includes: a sample
loading zone located upstream of a detection zone; a reporting
carrier zone located between the sample loading zone and a
detection zone, wherein said reporting carrier zone comprises a
reporting carrier capable of forming a complex with the analyte
said reporting carrier comprising a carrier and one or more
proficient enzyme cassettes; and a detection zone, wherein the
detection zone comprises a capture component for the analyte and an
indicator, wherein the indicator detects a reaction of a substrate
in the presence of a proficient enzyme thereby detecting the
presence of the analyte.
32. The lateral flow assay device of claim 31 further comprising a
control zone, wherein the control zone comprises a capture
component for the reporting carrier and an indicator for detecting
a reaction of a substrate in the presence of a proficient enzyme;
wherein the product of the enzyme reporting carrier complex and
substrate is detected thereby detecting the presence of the
reporting carrier.
33. The lateral flow assay device of claim 31 wherein the
proficient enzyme conjugate comprises an antibody.
34. The lateral flow assay device of claim 31 wherein the
proficient enzyme conjugate comprises a nucleic acid.
35. The lateral flow assay device of claim 31 wherein the
proficient enzyme conjugate comprises a proficient enzyme selected
from the group consisting of: urease, phosphocholine phosphatase,
betagalactosidase, xylose reductase, shikimate dehydrogenase,
malate dehydrogenase, carboxylesterase, neopullulanase, subtilisin,
4-phytase, acetylcholinesterase, laccase, bacterial leucyl
aminopeptidase, tripeptidyl-peptidase I, coagulation factor VIIa,
trypsin, betafructofuranosidase.
36. The lateral flow assay device of claim 31 wherein the antibody
interacts non-covalently with a proficient enzyme to form the
proficient enzyme conjugate.
37. The lateral flow assay device of claim 31 wherein the nucleic
acid is covalently attached to a proficient enzyme to form the
proficient enzyme conjugate.
38. The lateral flow assay device of claim 31 further providing one
or more inactive pro-enzymes in the detection zone.
39. The lateral flow assay device of claim 31, wherein the activity
of the proficient enzyme conjugate is detected by monitoring the
effect of the proficient enzyme assisted reaction.
40. The lateral flow assay device of claim 31, wherein the
indicator is a pH sensitive indicator.
41. The lateral flow assay device of claim 31, wherein the
indicator is a pH sensitive indicator is Potassium
I-hydroxy-4-[1-(2-hydroxyethylsulphonyl)phenylazo]-naphthalene-2-sulphona-
te.
42. The lateral flow assay device of claim 31, wherein the
indicator is a pH sensitive indicator is a pH sensitive cellulose
acetate coupled dye.
43. The lateral flow assay device of claim 31, wherein the
reporting carrier zone comprises a conjugate pad.
44. The lateral flow assay device of claim 31, wherein the sample
loading zone comprises a sample loading pad.
45. The lateral flow assay device of claim 31, further comprising a
rigid or flexible backing material.
46. The lateral flow assay device of claim 31, wherein the antibody
is associated with the proficient enzyme by noncovalent
interactions.
47. The lateral flow assay device of claim 31, wherein the antibody
or nucleic acid is covalently attached to the proficient
enzyme.
48. The lateral flow assay device of claim 31, further comprising a
source of one or more inactive pro-enzymes.
49. The lateral flow assay device of claim 31, wherein the
detection zone detects a product of the proficient enzyme by
detection of a pH change.
50. The lateral flow assay device of claim 31, wherein the pH
change is determined using a pH sensitive hydrogel.
51. The lateral flow assay device of claim 31, wherein the
detection zone detects the product of the proficient enzyme by
colorimetric change.
52. The lateral flow assay device of claim 31, wherein the
detection zone detects the product of the proficient enzyme by
fluorescence emission.
53. The lateral flow assay device of claim 31, wherein the
detection zone detects the product of the proficient enzyme by
electrochemical methods.
54. The lateral flow assay device of claim 51, wherein the
colorimetric change is due to silver ion reduction.
55. The lateral flow assay device of claim 31, the detection zone
detects the product of the proficient enzyme by precipitation of a
soluble component.
56. The lateral flow assay device of claim 31, wherein the soluble
component is a protein or pH sensitive polymer.
57. The lateral flow assay device of claim 56, wherein the protein
is BSA.
58. The lateral flow assay device of claim 56, wherein the pH
sensitive polymer is selected from the group consisting of methyl
acrylic acid, methyl methacrylate, methacrylic acid
2-(dimethylamino) ethyl ester, and N-hydroxymethyl acrylamide.
59. The lateral flow assay device of claim 31 further comprising an
absorptive pad at the distal end of the chromatographic medium.
60. The lateral flow assay device of claim 31, wherein the
substrate is allyl hexanoate and proficient enzyme is
carboxylesterase.
61. A lateral flow assay kit for detecting the presence of an
analyte within a test sample, the lateral flow assay device
comprising: a porous membrane comprising: a sample loading zone; a
reporting carrier zone down stream of the loading zone, wherein
said reporting carrier zone comprises a reporting carrier capable
of forming a complex with the analyte; a detection zone down stream
of the reporting carrier zone, wherein the detection zone comprises
a capture component and an indicator; and a substrate for the
proficient enzyme; wherein the substrate is applied to the
detection zone after the test sample has been allowed to flow
through the lateral flow device and the product of the enzyme and
substrate is detected.
62. The lateral flow assay kit of claim 61 further comprising a
control zone, wherein the control zone comprises a capture
component for the reporting carrier and an indicator for detecting
a reaction of a substrate in the presence of a proficient enzyme;
wherein the product of the enzyme reporting carrier complex and
substrate is detected thereby detecting the presence of the
reporting carrier.
63. The lateral flow assay kit of claim 61 wherein the proficient
enzyme conjugate comprises an antibody.
64. The lateral flow assay kit of claim 61 wherein the proficient
enzyme conjugate comprises a nucleic acid.
65. The lateral flow assay kit of claim 61 wherein the proficient
enzyme conjugate comprises a proficient enzyme selected from the
group consisting of: urease, phosphocholine phosphatase,
betagalactosidase, xylose reductase, shikimate dehydrogenase,
malate dehydrogenase, carboxylesterase, neopullulanase, subtilisin,
4-phytase, acetylcholinesterase, laccase, bacterial leucyl
aminopeptidase, tripeptidyl-peptidase I, coagulation factor Vita,
trypsin, betafructofuranosidase.
66. The lateral flow assay kit of claim 61 wherein the antibody
interacts non-covalently with a proficient enzyme to form the
proficient enzyme conjugate.
67. The lateral flow assay kit of claim 61 wherein the nucleic acid
is covalently attached to a proficient enzyme to form the
proficient enzyme conjugate.
68. The lateral flow assay kit of claim 61 further providing one or
more inactive pro-enzymes in the detection zone.
69. The lateral flow assay kit of claim 61, wherein the activity of
the proficient enzyme conjugate is detected by monitoring the
effect of the proficient enzyme assisted reaction.
70. The lateral flow assay kit of claim 61, wherein the indicator
is a pH sensitive indicator.
71. The lateral flow assay kit of claim 70, wherein the indicator
is a pH sensitive indicator is Potassium
I-hydroxy-4-[1-(2-hydroxyethylsulphonyl)phenylazo]-naphthalene-2-sulphona-
te.
72. The lateral flow assay kit of claim 70, wherein the indicator
is a pH sensitive cellulose acetate coupled dye.
73. The lateral flow assay kit of claim 61, wherein the reporting
carrier zone comprises a conjugate pad.
74. The lateral flow assay kit of claim 61, wherein the sample
loading zone comprises a sample loading pad.
75. The lateral flow assay kit of claim 61, further comprising a
rigid or flexible backing material.
76. The lateral flow assay kit of claim 61, wherein the antibody is
associated with the proficient enzyme by noncovalent
interactions.
77. The lateral flow assay kit of claim 61, wherein the antibody or
nucleic acid is covalently attached to the proficient enzyme.
78. The lateral flow assay kit of claim 61, further comprising a
source of one or more inactive pro-enzymes.
79. The lateral flow assay kit of claim 61, wherein the detection
zone detects a product of the proficient enzyme by detection of a
pH change.
80. The lateral flow assay kit of claim 79, wherein the pH change
is determined using a pH sensitive hydrogel.
81. The lateral flow assay kit of claim 61, wherein the detection
zone detects the product of the proficient enzyme by colorimetric
change.
82. The lateral flow assay kit of claim 61, wherein the detection
zone detects the product of the proficient enzyme by fluorescence
emission.
83. The lateral flow assay kit of claim 61, wherein the detection
zone detects the product of the proficient enzyme by
electrochemical methods.
84. The lateral flow assay kit of claim 81, wherein the
colorimetric change is due to silver ion reduction.
83. The lateral flow assay device of claim 61, the detection zone
detects the product of the proficient enzyme by precipitation of a
soluble component.
84. The lateral flow assay device of claim 83, wherein the soluble
component is a protein or pH sensitive polymer.
85. The lateral flow assay device of claim 84, wherein the protein
is BSA.
86. The lateral flow assay device of claim 84, wherein the pH
sensitive polymer is selected from the group consisting of methyl
acrylic acid, methyl methacrylate, methacrylic acid
2-(dimethylamino) ethyl ester, and N-hydroxymethyl acrylamide.
87. The lateral flow assay device of claim 60 further comprising an
absorptive pad at the distal end of the chromatographic medium.
88. The lateral flow assay device of claim 60, wherein the
substrate is allyl hexanoate and proficient enzyme is
carboxylesterase.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority of U.S. Provisional Patent
Application Ser. No. 61/535,874, entitled "MOLECULAR DIAGNOSTIC
ASSAY," filed on Sep. 16, 2011, which is incorporated by reference
herein.
FIELD OF THE INVENTION
[0002] The invention relates to the general field of molecular
biology, molecular diagnostics, nucleic acid testing (NAT), medical
science and biotechnology. The invention is suitable for detection
or monitoring traces of chemical and/or biological target(s) that
exist(s) in a very low concentration in samples, including, not
exclusively, biological samples, materials, organic or inorganic
samples. The traces of chemical and/or biological target(s) can
include biological/chemical products, fragments or whole target(s),
e.g. nucleic acid sequences, cells, viruses, pathogens, chemicals,
with applications in point of care/site/interest and laboratory in
fields such as pharmacogenomics, pathogen detection and monitoring,
determination of genetic predisposition, genetic classification for
clinical trials, diagnostics, prognostics, infectious disease
diagnostics and monitoring, bio-defense, forensic analysis,
paternity testing, animal and plant breeding, food testing, human
identification, genetically modified organism testing, chemical
contamination, food safety, monitoring and tracking in production
chain, and production in-line monitoring/control.
BACKGROUND
[0003] Molecular diagnostics has been a routine clinical laboratory
procedure for biological detection and monitoring. A specimen of a
biological sample coming from various biological sources usually
contains a mixture of chemicals, metabolites, macromolecules,
cells, virions, organisms, and nucleic acid sequences. Typically,
two common challenges exist for detection and monitoring:
background interference and limits of detection (LOD). Namely, the
target biological samples are usually present in a very small
amount compared with other background components in the biological
sample. Such non-target background components may interfere with
the detection downstream. LOD becomes an issue when not enough
copies of the target exist in the sample. The same LOD challenge is
the same as in chemical analysis, which usually requires highly
sophisticated instruments for conducting analysis work. Examples
include using mass spectrometry for detection of
bis(n-butyl)phthalate plasticizer contamination in the food supply
chain, melamine contamination in milk, cadmium and heavy metal
contamination in the soil or food. The background interference is
an order of magnitude higher than the contaminants. On-site
analysis is usually difficult without extensive treatment in the
laboratory involving instruments such as a GC spectrometer or mass
spectrometer, for example.
[0004] Background interference is usually solved by purifying the
sample during the preparation stage. Targets are captured and
background components are removed by one or more washing
procedure(s). Typical examples include: Enzyme-linked Immunosorbent
Assay (ELISA) and different chromatographic methods. Lateral flow
platforms can also be used to capture targets while background
components are removed from the immobilized targets. Another
example of a purification procedure for nucleic acid targets are
purification kits such as the Qiagen QIAamp DSP DNA Blood Mini
Kit.
[0005] The LOD issue for nucleic acids is usually resolved by
amplification of the sample target first, which allows later
detection by fluorescence emission, electrical and electronic
methods, e.g. voltametry, current measurement, capacitance
measurement, or impedance spectroscopy. Among these detection or
amplification methods, electrical power is required to provide the
light or conduct the electrical/electronic detection. One example
of resolving the LOD issue with low abundance nucleic acid targets
is by amplifying nucleic acid targets in vitro by using the
polymerase-chain reaction (PCR). The signal associated with the
presence of the target sequence can be further amplified by
fluorescence methods. Each PCR amplicon that is labeled by
fluorescent tags(s) can produce 1000 or 10,000 more protons in a
single excitation/measurement period when a fluorescence beacon or
probe is used to detect the amplicon. Another example is a
Campylobacter-like organism test, in which H. pylori cells
multiplies in the culture and is thus amplified, and where urease
secreted from the amplified cells can be detected after the number
of cells passes the detection threshold. Yet another example is the
screening of MRSA. This detection method differentiates the
colonies of the multiplied and amplified cells on a culture
medium.
[0006] However, current methods which rely on removal of various
combinations of background interference and amplification detection
have not filled the need for a robust, cost effective, rapid, easy
to use detection method for targets, which is compatible in
resource limited environments. The requirement for an electrical
power source to perform the current tests by powering the
instrument or providing the temperature incubation has restricted
access to this technology in resource-poor regions or conditions.
The time required to practice the current methods is long. Many
hours or days are required to produce enough biological targets to
generate sufficient detection signals. The amplification step used
for pathogen detection, metabolite determination, or nucleic acid
sequence detection usually requires expensive laboratory
instruments and trained specialists to run the instruments. In the
case of PCR, care is required to handle labile reagents, and
special diligence is essential to avoid contamination between
samples. Moreover, the instruments required to perform PCR are
expensive and complex. The above considerations are severe
limitations that prevent POC (point of care) use or provide rapid
sample-to-result in less than 30 minutes. The present invention
provides solutions to these and other needs.
SUMMARY
[0007] Methods and devices for molecular detection or diagnostic
assays are provided by the present invention. The methods disclosed
herein are suitable for detection or monitoring of one or more
targets that exist in very low concentrations in samples including
biological, chemical and material but not exclusively limited to
these, and generally allow detection in the absence of the
replication of the target or amplification of a fragment or part of
the target. The methods and devices of the present application are
amenable to point of care detection without the requirement for
access to electrical power.
[0008] In one aspect, the present invention provides a method for
detecting an analyte by performing the steps of i) providing a
lateral flow assay device that comprises a chromatographic medium
that includes: (a) a sample loading zone located upstream of a
detection zone; (b) a reporting carrier zone located between the
sample loading zone and a detection zone, wherein said reporting
carrier zone comprises a reporting carrier capable of forming a
complex with the analyte said reporting carrier comprising a
carrier and one or more proficient enzyme cassettes; and (c) a
detection zone, wherein the detection zone comprises a capture
component for the analyte and an indicator; ii) Contacting the
sample application zone with the test sample, wherein the test
sample travels through the reporting carrier zone along the
chromatographic medium from the sample loading zone to the
detection zone and beyond the detection zone; iii) adding a
substrate to the detection zone wherein the substrate undergoes a
reaction in the presence of proficient enzyme analyte containing
reporting carrier; and iv) generating a response of the indicator
within the detection zone that corresponds to the presence or
absence of the analyte in the test sample.
[0009] In another aspect, the present invention provides a device
for detecting an analyte by comprising: a chromatographic medium
that includes: a sample loading zone located upstream of a
detection zone; a reporting carrier zone located between the sample
loading zone and a detection zone, wherein said reporting carrier
zone comprises a reporting carrier capable of forming a complex
with the analyte said reporting carrier comprising a carrier and
one or more proficient enzyme cassettes; and a detection zone,
wherein the detection zone comprises a capture component for the
analyte and an indicator wherein the indicator detects a reaction
of a substrate in the presence of a proficient enzyme thereby
detecting the product of the analyte enzyme reporting carrier
complex and a substrate is detected, thereby detecting the presence
of the analyte.
[0010] In one aspect, the present invention provides a kit for
detecting an analyte comprising a lateral flow assay device
comprising: a porous membrane comprising: a sample loading zone; a
reporting carrier zone down stream of the loading zone, wherein
said reporting carrier zone comprises a reporting carrier capable
of forming a complex with the analyte; a detection zone down stream
of the reporting carrier zone, wherein the detection zone comprises
a capture component and an indicator; and a substrate for the
proficient enzyme; wherein the substrate is applied to the
detection zone after the test sample has been allowed to flow
through the lateral flow device and the product of the enzyme and
substrate is detected.
[0011] In some embodiments, it is also possible to implement the
method comprising these steps, but not in the order listed above.
For example, it is possible to (a) immobilize the analyte from a
sample suspected of containing the analyte (b) contacting the
immobilized analyte to a reporting carrier to form a reporting
carrier-analyte complex, where the reporting complex comprises a
proficient enzyme, (c) providing a substrate for the proficient
enzyme, and (d) detecting fluctuation as a result of proficient
enzyme assisted reactions, for example, product of the reaction,
thereby detecting the presence of the analyte.
[0012] In various embodiments of this aspect, the analyte is a
protein, nucleic acid cell, part of a cell or pathogen, pathogen,
virion, component of cellular or extracellular matrix, or small
molecules. In various embodiments of this aspect, the reporter
carrier contains an antibody or nucleic acid.
[0013] In certain embodiments of this aspect, the proficient enzyme
can be urease, phosphocholine phosphatase, beta-galactosidase,
xylose reductase, shikimate dehydrogenase, malate dehydrogenase,
neopullulanase, subtilisin, 4-phytase, acetylcholinesterase,
laccase, bacterial leucyl aminopeptidase, tripeptidyl-peptidase I,
coagulation factor VIIa, trypsin, betafructofuranosidase.
[0014] In an embodiment of this aspect, the antibody is associated
with the proficient enzyme by noncovalent interactions.
Alternatively, the antibody or nucleic acid is covalently attached
to the proficient enzyme.
[0015] In an additional embodiment of this aspect, the method
further uses one or more inactive pro-enzymes. A pro-enzyme is any
of a group of compounds that are inactive precursors of enzymes and
require some change (such as the hydrolysis of a fragment that
masks an active enzyme) to become active.
[0016] In another embodiment of this aspect, the detection of the
product of the proficient enzyme is by detection of a pH change,
which may be determined using a pH sensitive hydrogel that can have
gold nanoparticles that change color in response to pH changes.
[0017] In a further embodiment of this aspect, the detection of the
product of the proficient enzyme is by colorimetric change, where
the colorimetric change is due to fluctuations as a result of
proficient enzyme assisted reaction, for example, protonation or
deprotonation of the dye material or silver ion reduction.
[0018] In yet further embodiments of this aspect, the detection of
the product of the proficient enzyme is by precipitation as the
result of the effect of the proficient enzyme-assisted reaction,
for example, precipitation of a soluble component, which can be a
protein including BSA or pH sensitive polymer, which forms
aggregates with changes in pH. The change of pH results in the
shift of the surface charge of the polymer. As a consequence of the
surface charge variation, the 3-D structure of the polymer
molecules changes and the hydrophobic part of the polymer is
exposed. The exposure of the hydrophobic region of the polymer
increases the entropy. These hydrophobic parts of the polymer would
form noncovalent interactions thus forming aggregates. Examples of
pH sensitive polymers include: methyl acrylic acid, methyl
methacrylate, methacrylic acid 2-(dimethylamino) ethyl ester, and
N-hydroxymethyl acrylamide.
[0019] In another aspect, the present invention provides a
molecular diagnostic device that has (a) a sample loading zone, (b)
a reporting carrier zone, where the reporting carrier zone contains
a reporting carrier and a proficient enzyme, (c) a source of
substrate for the proficient enzyme; and (d) a detection zone,
where the detection zone contains a capture component.
[0020] In some embodiments of this aspect, the device also has a
positive control zone.
[0021] In some embodiments of this aspect, the sample loading zone
is a sample loading pad.
[0022] In some embodiments of this aspect, the reporting carrier
zone is a conjugate pad.
[0023] In some embodiments of this aspect, the detection area
comprises a porous membrane.
[0024] In some embodiments of this aspect, the device has a rigid
or flexible backing material.
[0025] In various embodiments of this aspect, the reporter carrier
contains an antibody or nucleic acid.
[0026] In certain embodiments of this aspect, the proficient enzyme
can be urease, phosphocholine phosphatase, beta-galactosidase,
xylose reductase, shikimate dehydrogenase, malate dehydrogenase,
neopullulanase, carboxylesterase, subtilisin, 4-phytase,
acetylcholinesterase, laccase, bacterial leucyl aminopeptidase,
tripeptidyl-peptidase I, coagulation factor VIIa, trypsin,
betafructofuranosidase. In certain embodiments of the present
invention when a pro-enzyme it utilized, the pro-enzyme may be a
pro-enzyme selected from the group of pro-enzymes of the above
proficient enzymes.
[0027] In an embodiment of this aspect, the antibody is associated
with the proficient enzyme by noncovalent interactions.
Alternatively, the antibody or nucleic acid is covalently attached
to the proficient enzyme.
[0028] In an additional embodiment of this aspect, the method
further uses one or more inactive pro-enzymes.
[0029] In another embodiment of this aspect, the detection of the
product of the proficient enzyme is by detection of a pH change,
which may be determined using a pH sensitive hydrogel that can have
gold nanoparticles that change color in response to pH changes.
[0030] In a further embodiment of this aspect, the detection of the
product of the proficient enzyme is by colorimetric change, where
the colorimetric change is due to the effect of a proficient enzyme
assisted reaction, for example, protonation or deportonation of the
dye material or silver ion reduction.
[0031] In yet further embodiments of this aspect, the detection of
the product of the proficient enzyme is by precipitation as the
result of the fluctuation of the proficient enzyme-assisted
reaction, for example, precipitation of a soluble component, which
can be a protein including BSA or pH sensitive polymer. Examples of
pH sensitive polymers include: methyl acrylic acid, methyl
methacrylate, methacrylic acid 2-(dimethylamino) ethyl ester, and
N-hydroxymethyl acrylamide.
[0032] In another further aspect, the present invention provides a
molecular diagnostic device that has (a) a sample loading zone, (b)
a pre-reporting carrier zone, if there is one, where the
pre-reporting carrier zone contains a reporting carrier and a
proficient enzyme, (c) a source of substrate for the proficient
enzyme and a reporting carrier for the pre-reporting carrier; and
(d) a detection zone, where the detection zone contains a capture
component.
[0033] In another further aspect, the present invention provides a
molecular diagnostic device that has a detection zone, where the
detection zone contains a capture component. The pre-reporting
carrier, reporting carrier, or substrate could be added to the
detection sequentially.
[0034] In another further aspect, the present invention provides a
molecular diagnostic device that has a detection zone, where the
detection zone contains a capture component. The detection zone
could be placed and moved amongst multiple reagent containers that
contain one or more of the following: pre-reporting carrier,
reporting carrier, and substrate.
[0035] In yet another further aspect, the present invention
provides a molecular diagnostic device that utilizes combinations
of the process highlighted in paragraphs [0032], [0033], and
[0034].
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] FIG. 1 shows general steps for target detection, including
sample preparation, signal carrier attachment, signal carrier
immobilization, excess molecule removal, and result display.
[0037] FIG. 2 shows the general steps for nucleic acid
detection.
[0038] FIG. 3 shows implementation on a lateral flow device.
[0039] FIG. 4A-4H shows various embodiments of a lateral flow
device.
[0040] FIG. 5A-5L shows various embodiments of the function of a
lateral flow device.
[0041] FIG. 6A-6H shows different embodiments of pre-reporter
carriers for the detection of nucleic acid targets.
[0042] FIG. 7 illustrates a lateral flow device with a positive
test result.
[0043] FIG. 8 illustrates a lateral flow device with a negative
test result.
DETAILED DESCRIPTION
[0044] The present invention generally provides methods and devices
for the detection of analytes and targets in a sample with a
sufficiently low limit of detection (LOD) to reduce or eliminate
the need for an amplification step prior to detection. The methods
disclosed herein provide a significantly less complex process with
a reduced requirement for sample purity. The methods and devices of
the present invention also provide a streamlined sample preparation
process, including for sample enrichment, purification, labeling,
and detection.
[0045] A lateral flow molecular diagnostic device for detecting the
presence of an analyte in a test sample comprising a
chromatographic medium that includes: (a) a sample loading zone
located upstream of a detection zone; (b) a reporting carrier zone
located between the sample loading zone and a detection zone,
wherein said reporting carrier zone comprises a reporting carrier
capable of forming a complex with the analyte; (c) a substrate for
the proficient enzyme; and (d) a detection zone, wherein the
detection zone comprises a capture component for the analyte and a
substrate for the proficient enzyme; wherein the product of the
enzyme and substrate is detected.
[0046] The lateral flow diagnostic device for detecting the
presence of an analyte in a test sample may further comprise a
positive control zone downstream of the detection zone.
[0047] Attempts at development of nucleic acid detection devices
for use in the near-bedside environment have been made. Some
products have partially filled this need by bringing the test
outside the clinical laboratory: However, these approaches do not
(1) eliminate the reliance on an amplification step and associated
equipment, (2) reduce the storage requirement for detection
materials, (3) eliminate the reliance on a complex analyzer that
reads an optical signal from the amplification; or (4) eliminate
the reliance on an electrical power supply. The amplified nucleic
acid fragment as the result of the said nucleic acid amplification
test has fundamentally increased the risk of cross-contamination
between samples.
[0048] Lateral flow devices that seek to replace the need for a
scanner or analyzer have also be described in, for example, US
2011086359, WO2004092342, Anal. Chem, 2004, V76, P888, and Anal.
Chem. 2009, V81, P1660. However, these methods, while not requiring
a complex analyzer, suffer from a lack of sensitivity for direct
detection without pre-amplification. For example, pathogen nucleic
acid detection generally requires 1,000 copies or better to achieve
sufficient sensitivity; other reports describe a requirement for a
minimum of 100,000,000 copies of amplified targets for detection.
These methods have a requirement for amplification instruments and
reagents, which are sensitive to storage condition; require
specialists to perform the amplification of the target nucleic
acids; and are sensitive to cross contamination through the
repeated use of the same amplification instruments.
[0049] There are also rapid kits for detecting presence of a
pathogen, such as CLO
(http://www.medicinenet.com/helicobacter_pylori/article.htm#2diagnosis),
but these methods require (1) long incubation time (from a few
hours to days) to multiply biological targets; (2) specialists to
take a biopsy; and (3) specialist to interpret the result.
[0050] As described in detail herein, the present invention
provides methods and devices to detect one or more targets,
including biological, chemical, or material targets with minimal or
no amplification prior to the detection. In part, this is
accomplished through the use of stable and robust proficient enzyme
systems that allow direct detection of a biological target without
pre-amplification. In the case of nucleic acid detection, there is
reduced risk of cross contamination because the target is not
replicated and the entire device is disposable. Detection time is
much reduced, with sample-to-result times of less than 1 hour or as
short as 15 minutes. The enzymes used in the present invention are
preferably stable at room temperature. In some embodiments, the
invention enables detection without sophisticated instrumentation,
thus making the invention amenable to point of care (POC)
applications.
[0051] Accordingly, the methods and devices of the present
invention provide for significantly reduced setup costs and
equipment requirements for point of care detection and are amenable
for application to a disposable kit. These features allow for
embodiments which can be operated directly by consumers or other
personnel without prior training before use. Furthermore, the
methods and devices of the present invention can be integrated into
existing workflows to improve limit of detection (LOD) and/or
detection time.
DEFINITIONS
[0052] It is to be understood that this invention is not limited to
particular methods, reagents, compounds, compositions or biological
systems, which can, of course, vary. It is also to be understood
that the terminology used herein is for the purpose of describing
particular aspects only, and is not intended to be limiting. As
used in this specification and the appended claims, the singular
forms "a", "an" and "the" include plural references unless the
content clearly dictates otherwise.
[0053] The term "about" as used herein when referring to a
measurable value such as an amount, a temporal duration, and the
like, is meant to encompass variations of .+-.20% or .+-.10%, more
preferably .+-.5%, even more preferably .+-.1%, and still more
preferably .+-.0.1% from the specified value, as such variations
are appropriate to perform the disclosed methods.
[0054] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the invention pertains. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice for testing of the present
invention, the preferred materials and methods are described
herein.
[0055] An "analyte" or "target" refers to a compound to be
detected. Such compounds can include cells, parts of cells or
pathogens, pathogens, virions, components of cellular or
extracellular matrix, small molecules, peptides, proteins, nucleic
acids, as well as other chemical entities.
[0056] Chromatographic medium may be made from any of a variety of
materials through which the test sample is capable of passing. For
example, the chromatographic medium may be a porous membrane formed
from synthetic or naturally occurring materials, such as
polysaccharides (e.g., cellulose materials such as paper and
cellulose derivatives, such as cellulose acetate and
nitrocellulose); polyether sulfone; polyethylene; nylon;
polyvinylidene fluoride (PVDF); polyester; polypropylene; silica;
inorganic materials, such as deactivated alumina, diatomaceous
earth, MgSO.sub.4, or other inorganic finely divided material
uniformly dispersed in a porous polymer matrix, with polymers such
as vinyl chloride, vinyl chloride-propylene copolymer, and vinyl
chloride-vinyl acetate copolymer; cloth, both naturally occurring
(e.g., cotton) and synthetic (e.g., nylon or rayon); porous gels,
such as silica gel, agarose, dextran, and gelatin; polymeric films,
such as polyacrylamide; and so forth. In one particular embodiment,
the chromatographic medium is formed from nitrocellulose and/or
polyether sulfone materials. It should be understood that the term
"nitrocellulose" refers to nitric acid esters of cellulose, which
may be nitrocellulose alone, or a mixed ester of nitric acid and
other acids, such as aliphatic carboxylic acids having from 1 to 7
carbon atoms.
[0057] The size and shape of the chromatographic medium may
generally vary as is readily recognized by those skilled in the
art. For instance, a porous membrane strip may have a length of
from about 10 to about 100 millimeters, in some embodiments from
about 20 to about 80 millimeters, and in some embodiments, from
about 40 to about 60 millimeters. The width of the membrane strip
may also range from about 0.5 to about 20 millimeters, in some
embodiments from about 1 to about 15 millimeters, and in some
embodiments, from about 2 to about 10 millimeters. Likewise, the
thickness of the membrane strip is generally small enough to allow
transmission-based detection. For example, the membrane strip may
have a thickness less than about 500 micrometers, in some
embodiments less than about 250 micrometers, and in some
embodiments, less than about 150 micrometers.
[0058] As stated above, the support material carries the
chromatographic medium. For example, the support 21 may be
positioned directly adjacent to the chromatographic medium as shown
in FIG. 3, or one or more intervening layers may be positioned
between the chromatographic medium and the support material.
Regardless, the support material may generally be formed from any
material able to carry the chromatographic medium. The support
material may be formed from a material that is transmissive to
light, such as transparent or optically diffuse (e.g.,
transluscent) materials. Also, it is generally desired that the
support material is liquid-impermeable so that fluid flowing
through the medium does not leak through the support material.
Examples of suitable materials for the support include, but are not
limited to, glass; polymeric materials, such as polystyrene,
polypropylene, polyester (e.g., Mylar.RTM. film), polybutadiene,
polyvinylchloride, polyamide, polycarbonate, epoxides,
methacrylates, and polymelamine; and so forth. To provide a
sufficient structural backing for the chromatographic medium, the
support material is generally selected to have a certain minimum
thickness. Likewise, the thickness of the support 8 is typically
not so large as to adversely affect its optical properties. Thus,
for example, the support material may have a thickness that ranges
from about 100 to about 5,000 micrometers, in some embodiments from
about 150 to about 2,000 micrometers, and in some embodiments, from
about 250 to about 1,000 micrometers.
[0059] As is well known the art, the chromatographic medium may be
cast onto the support material wherein the resulting laminate may
be die-cut to the desired size and shape. Alternatively, the
chromatographic medium may simply be laminated to the support
material with, for example, an adhesive. In some embodiments, a
nitrocellulose or nylon porous membrane is adhered to a film.
[0060] The lateral flow device also contains an absorbent material
that is positioned adjacent to the chromatographic medium at the
distal end, That is the end of the chromatographic medium farthest
from the sample loading zone. The absorbent material assists in
promoting capillary action and fluid flow through the
chromatographic medium. In addition, the absorbent material
receives fluid that has migrated through the entire chromatographic
medium and thus draws any unreacted components away from the
detection and control regions to help reduce the likelihood of
"false positives." Some suitable absorbent materials that may be
used in the present invention include, but are not limited to,
nitrocellulose, cellulosic materials, porous polyethylene pads,
glass fiber filter paper, and so forth. The absorbent material may
be wet or dry prior to being incorporated into the device.
Pre-wetting may facilitate capillary flow for some fluids, but is
not typically required. Also, as is well known in the art, the
absorbent material may be treated with a surfactant to assist the
wicking process.
[0061] The sample loading zone may be formed by a separate
material, such as a pad. Some suitable materials that may be used
to form such sample pads include, but are not limited to,
nitrocellulose, cellulose, porous polyethylene pads, and glass
fiber filter paper. If desired, the sample loading zone may also
contain one or more pretreatment reagents, either diffusively or
non-diffusively attached thereto. In the illustrated embodiment,
the test sample travels from the sample loading zone to a reporting
carrier zone that is in communication with the sample loading zone.
The reporting carrier zone may be formed on the chromatographic
medium. Alternatively, as shown in FIG. 3, the reporting carrier
zone is formed from a separate material or pad. Such a reagent pad
may be formed from any material through which the test sample is
capable of passing, such as glass fibers.
[0062] An "indicator" refers to any of various substances, such as
litmus, phenolphthalein, or bromothymol blue, Potassium
I-hydroxy-4-[1-(2-hydroxyethylsulphonyl)phenylazo]-naphthalene-2-sulphona-
te, cellulose acetate coupled potassium
I-hydroxy-4-[1-(2-hydroxyethylsulphonyl)phenylazo]-naphthalene-2-sulphona-
te and the like that indicate the presence, absence, or
concentration of another substance or the degree of reaction
between two or more substances by means of a characteristic change,
especially in color.
[0063] A "sample" refers to any source which is suspected of
containing an analyte or target molecule. Examples of samples which
may be tested using the present invention include, but are not
limited to, blood, serum, plasma, urine, saliva, cerebrospinal
fluid, lymph fluids, tissue and tissue and cell extracts, cell
culture supernatants, biopsy specimens, paraffin embedded tissue,
soil, fruit, juice, oil, milk, food, water, among others. A sample
can be suspended or dissolved in liquid materials such as buffers,
extractants, solvents, and the like.
[0064] "Reporting carrier" refers to an entity which binds and
forms a complex with a target or analyte and reports the presence
of the target or analyte. The reporting carrier can comprise a
protein or nucleic acid or another moiety that is able to form a
complex with an analyte or target molecule. The reporting carrier
is associated with a proficient enzyme cassette, which can comprise
combinations of enzymes and substrates, which are only active in
the presence of a target or analyte. The enzyme cassette contains
at least one proficient enzyme that is conjugated to a reporting
carrier.
[0065] In some embodiments, the carrier is an antibody or
oligonucleotide.
[0066] "Proficient enzyme" or "high yield enzyme" refers to an
enzyme that can generate a product at a high rate that approaches
the diffusion limit.
[0067] "Capture component" refers generally to a molecule that
specifically recognizes and complexes with a target or analyte
without preventing a reporting carrier from forming a complex with
the same target or analyte. Generally, the capture component is
immobilized to a matrix on the chromatographic membrane.
[0068] "Pre-reporting carrier" refers generally to a molecule that
specifically binds to the analyte before the association of
reporting carrier. A pre-reporting carrier generally does not
contain a proficient enzyme.
[0069] A "proficient enzyme conjugate" refers generally to a
proficient enzyme, which is conjugated to a reporting carrier. The
nature of the interaction is covalent or non-covalent or a hybrid
of both.
Components and Methods
Reporting Carrier
A. Carriers
[0070] A reporting carrier typically comprises two components: the
first component is a carrier which is able to form a specific
complex with a target or analyte, which can be biological, chemical
or other types. The other component is one or more proficient
enzyme cassettes. Among the carriers that can be used in the
practice of the present invention include, but are not limited to,
nucleic acids with a sequence which is complementary to at least
part of a target nucleic acid sequence, antibodies, aptamers, solid
or porous microparticles, and synthetic polymer with an imprinted
structure that is complimentary to at least part of the target.
Solid, shell-core, or porous microparticles can be part of the
carrier, which can improve the control of the flow to direct the
carrier to the reaction pads. This can be achieved by changing the
size or density of the carrier or providing the utility of
magnet-driven particle movement, if particles can be polarized
magnetically, e.g. paramagnetic particles.
[0071] In another embodiment, a pre-reporting carrier, which does
not contain a proficient enzyme, forms a pre-reporting
carrier-analyte complex, said complex A. Complex A can be captured
and immobilized by an immobilizer. The pre-reporting carrier alone
does not bind with the immobilizer. Without the presence of the
analyte, there is no complex formation and the pre-reporting
carrier is washed away.
[0072] The reporting carrier is specific to the analyte. A
pre-reporting carrier could be used to form a complex with the
analyte. Such association is possible when all three components are
present in the solution. In a preferred embodiment, a pre-reporting
carrier is added to the sample solution prior to the sample
solution being applied to the lateral flow assay device. The
analyte pre-reporting carrier complex comes into contact in the
reporter carrier zone, wherein the reporting carrier forms an
association with the complex. The analyte is captured and
immobilised by the surface capture agent in the detection zone. The
association of the analyte and the pre-reporting carrier does not
inhibit the immobilisation of the analyte. In the presence of the
analyte and pre-reporting carrier, the complex of the said two
component would be immobilised via the interaction of the analyte
and the surface capture agent in the detection zone. The reporting
carrier is then allowed to be immobilised when it associated with
the complex. In the absence of analyte or pre-reporting carrier
analyte complex, the reporting carrier would not be able to be
immobilised and would move past the detection zone as the solvent
moves and optional washes move down the lateral flow device.
[0073] The purpose of the pre-reporting carrier is multiple. One is
to increase specificity to the target analyte. The conjugation of
the enzyme to the reporting carrier could restrict the ability to
maximize the binding selectivity to the target analyte over
background non-targets. In the case of nucleic acid, the steric
hindrance or coulomb charge effect from the conjugated enzyme could
affect he selectivity of the hybridization. The other purpose is to
increase the signal amplification process through cascade binding.
One example is the detection of a small analyte. When the molecular
size of the analyte is too small, it is difficult to have more than
one reporting carrier forming complex with one analyte. There is
more surface area per analyte molecule after it forms a complex,
said complex A, with the pre-reporting carrier. Larger surface area
and functional groups are created as a result of complex A
formation. The added surface area and functional groups would allow
more than one reporting carrier to be associated with the complex,
which comprise one analyte. More reporting carriers would produce
more signals and therefore, further improve the signal to noise
ratio. Using a pre-reporting carrier increases the target surface
area to allow more reporting carriers specific binding events per
analyte to take place.
[0074] Another embodiment reduces the complexity of the reporting
carrier design and increases multiplicity to the target analytes,
e.g. virus gene polymorphism or multiple nucleic acid sequence
targets. A mixture of the various pre-reporting carriers could be
designed to react with each specific genotype or sequence. All the
pre-reporting carriers could contain one or more tags that is/are
specific to the reporting carrier. When using the same single tag,
the reporting carrier binds to the tag specifically but without
differentiation between the pre-reporting carriers. When using
different tags, it is useful to group several targets separately if
a different reporting carrier is designed to each tag.
[0075] FIGS. 6A-6H show different examples of pre-reporting
carriers for use in nucleic acid target detection. For example, as
shown in FIGS. 6A and 6B, a pre-reporting carrier can comprise
unique target binding regions (e.g., I to N), each with a unique
tag (e.g., a1 to a3). Each of the unique tags would be bound by a
different reporting carrier. One advantage of this variation is
that the pre-reporting carriers (and thus, also, the corresponding
reporter carriers) bind at different target locations. This reduces
the risk of false positives resulting from mis-binding of the
pre-reporter or reporter carrier to the wrong reaction spots. FIG.
6C shows an example where unique target binding regions (I-N) each
carry the same tag ("a"). As shown in FIGS. 6D-6F, each
pre-reporter carrier could hybridize to part of the same target
with detection using a single species of reporter carrier bearing a
proficient enzyme conjugate. In further examples, each pre-reporter
carrier with the same tag ("a") can bind to different targets as
opposed to different parts of the same target (FIGS. 6F-6H).
[0076] "Antibody" refers to any immunoglobulin or intact molecule
as well as to fragments thereof that bind to a specific epitope
that may be used in the practice of the present invention. Such
antibodies include, but are not limited to polyclonal, monoclonal,
chimeric, humanized, single chain, Fab, Fab', F(ab)' fragments
and/or F(v) portions of the whole antibody and variants thereof.
All isotypes are encompassed by this term and may be used in the
practice of this invention, including IgA, IgD, IgE, IgG, and
IgM.
[0077] An "antibody fragment" refers specifically to an incomplete
or isolated portion of the full sequence of the antibody which
retains the antigen binding function of the parent antibody and may
also be used in the present invention. Examples of antibody
fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies;
linear antibodies; single-chain antibody molecules; and
multispecific antibodies formed from antibody fragments.
[0078] An intact "antibody" for use in the invention comprises at
least two heavy (H) chains and two light (L) chains inter-connected
by disulfide bonds. Each heavy chain is comprised of a heavy chain
variable region (abbreviated herein as HCVR or VH) and a heavy
chain constant region. The heavy chain constant region is comprised
of three domains, CH1, CH2 and CH3. Each light chain is comprised
of a light chain variable region (abbreviated herein as LCVR or VL)
and a light chain constant region. The light chain constant region
is comprised of one domain, CL. The VH and VL regions can be
further subdivided into regions of hypervariability, termed
complementarity determining regions (CDR), interspersed with
regions that are more conserved, termed framework regions (FR).
Each VH and VL is composed of three CDRs and four FRs, arranged
from amino-terminus to carboxyl-terminus in the following order:
FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the
heavy and light chains contain a binding domain that interacts with
an antigen. The constant regions of the antibodies can mediate the
binding of the immunoglobulin to host tissues or factors, including
various cells of the immune system (e.g., effector cells) and the
first component (Clq) of the classical complement system. The term
antibody includes antigen-binding portions of an intact antibody
that retain capacity to bind. Examples of binding include (i) a Fab
fragment, a monovalent fragment consisting of the VL, VH, CL and
CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment
comprising two Fab fragments linked by a disulfide bridge at the
hinge region; (iii) a Fd fragment consisting of the VH and CH1
domains; (iv) a Fv fragment consisting of the VL and VH domains of
a single arm of an antibody, (v) a dAb fragment (Ward et al.,
Nature, 341:544-546 (1989)), which consists of a VH domain; and
(vi) an isolated complementarity determining region (CDR).
[0079] "Single chain antibodies" or "single chain Fv (scFv)" may
also be used in the present invention. This term refers to an
antibody fusion molecule of the two domains of the Fv fragment, VL
and VH. Although the two domains of the Fv fragment, VL and VH, are
coded for by separate genes, they can be joined, using recombinant
methods, by a synthetic linker that enables them to be made as a
single protein chain in which the VL and VH regions pair to form
monovalent molecules (known as single chain Fv (scFv); see, e.g.,
Bird et al., Science, 242:423-426 (1988); and Huston et al., Proc
Natl Acad Sci USA, 85:5879-5883 (1988)). Such single chain
antibodies are included by reference to the term "antibody"
fragments can be prepared by recombinant techniques or enzymatic or
chemical cleavage of intact antibodies.
[0080] A "monoclonal antibody" may be used in the present
invention. Monoclonal antibodies are a preparation of antibody
molecules of single molecular composition. A monoclonal antibody
composition displays a single binding specificity and affinity for
a particular epitope.
[0081] Non-limiting examples of the specificity of antibodies that
may be used in the practice of the invention include those specific
for particular antigens; methylation specific antibodies for the
detection of methylated DNA; and phosphorylation specific
antibodies for the detection of phosphoproetins, among others. For
example, virus specific antibodies can be used to detect the
presence of virions in samples. All of the antibodies and fragments
described above can be modified with groups that allow an antibody
or fragment to be attached to a proficient enzyme.
[0082] In some embodiments, a nucleic acid is used as a carrier. If
a nucleic acid is used, an oligonucleotide with a sequence
complementary to at least part of a target nucleic acid can be
used. Methods for synthesizing nucleic acids are well known in the
art. Nucleic acids of the invention may be modified with groups
that allow the nucleic acid to be attached to a proficient enzyme.
Mismatches of one or a few selected bases of the carrier nucleic
acid to the target may be introduced. Utilizing the artificially
introduced mismatch(s), the binding energy could be adjusted to
design or engineer selectivity of the carrier over different
samples as the result of enthalpy and entropy change.
[0083] In addition to oligonucleotides comprising natural bases,
oligonucleotides comprising nucleotide analogues may be used, such
as peptide nucleic acids (PNAs) or locked nucleic acids (LNAs). In
contrast to DNA and RNA, the backbone of PNAs are composed of
repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. A
locked nucleic acid (LNA) is a modified RNA nucleotide. The ribose
moiety of an LNA nucleotide is modified with an extra bridge
connecting the 2' oxygen and 4' carbon. The bridge "locks" the
ribose in the 3'-endo (North) conformation, which is often found in
A-form duplexes. LNA nucleotides can be mixed with DNA or RNA
residues in an oligonucleotide as needed. Such oligomers can be
synthesized chemically and are commercially available.
B. Proficient Enzymes or Carrier Enzymes
[0084] The carriers described above can be associated noncovalently
or covalently with various proficient enzymes or carrier enzymes in
the practice of the invention. Enzymes particularly useful in the
practice of the present invention are characterized by reaction
rates which approach the diffusion limit of the substrate. A
proficient enzyme of the invention typically can have a turnover
constant of 10.sup.3 per second per enzyme to 10.sup.8 or higher
per second per enzyme.
[0085] Examples of suitable proficient enzymes or carrier enzymes
are included in the following EC classes, but are not limited to,
oxidoreductase (EC 1), hydrolase (EC3), lyases (EC4), or ligases
(EC6), or an enzyme, enzyme-like nucleic acid, or enzyme-like
catalyst that catalyzes a reaction that produces a proton
concentration.
[0086] Examples of such enzymes are known in the art. Examples of
enzymes useful in the practice of the present invention and their
characteristics and substrates include, but are not limited to,
those shown below.
1. Urease
EC Number: 3.5.1.5, Hydrolase
[0087] a. Turnover number: 2.97.times.10.sup.3 S.sup.-1 b.
Substrate: Urea
Ref 1: PDB: 1 fwj.
Ref 2: Pearson, M. A. et al, <Kinetic and Structural
Characterization of Urease Active Site Variants>Biochemistry,
2000, 39(29) p 8575-8584
[0088] 2. Phosphocholine phosphatase EC number: 3.1.3.75 aka
phosphoethanolamine Spec: Pseudomonas aeruginosa PA5292 gene in
PAO1 genome wild-type (construct with truncation and modification
in the plasmid construct) a. Turnover number: 5-7.times.10.sup.6
S.sup.-1 b. Substrate: p-nitrophenyl phosphate c. Product:
p-nitrophenol Ref 1: Beassoni, P. R. et al. Current Microbiology,
2006 52(6), p534-539
3. Beta-galactosidase
[0089] EC number: 3.2.1.23 from Kluyveromyces marxianus a. Turnover
number: 2.7.times.10.sup.6 S.sup.-1 b. Substrate:
4-nitrophenyl-beta-D-galactoside Ref 1: Oconnell, S. et. al.
Applied Biochemistry and Biotechnology 2007 V141(1) p1-13 4. Xylose
reductase EC number: 1.1.1.307 from Talaromyces emersonii a.
Turnover number: 1-3.times.10.sup.5 S.sup.-1 b. Substrate:
D-Xylose, NADPH Ref 1: Fernandes, S, et al. J. Biosci. 34(6) 2009
p881-890
5. Shikimate Dehydrogenase
[0090] EC 1.1.1.25 Escherichia coli Species: Wild type (shikimate),
Mutant S22A, Y39F, D107A, S67A, T106A a. Turnover number:
1.times.10.sup.5 S.sup.-1 b. Substrate: Shikimate, or quinate, or
NAD+ Ref 1: Lindner, H. A. J. Bio. Chem. 2004 V280, P 7162-7169 6.
Malate dehydrogenase EC number: 1.1.1.37 Triticum aestivum or
Talaromyces emersonii a. Turnover number: 1.times.10.sup.5 S.sup.-1
b. Substrate: NADH Ref 1: Maloney, A. P. et al. Eur. J. Biochem 271
2004 p 3115-3126
7. Neopullulanase
[0091] EC number: 3.2.1.135 from Geobacillus stearothermophilus a.
Turnover number: 1.times.10.sup.5 S.sup.-1 b. Substrate: Starch
(Pullulan film aka E1204) Ref 1: Zareian, S et al., Enzyme Microb.
Technol. 2010 46 p57-63
8. Subtilisin
[0092] EC number: 3.4.21.62 from Bacillus sp. a. Turnover number:
1.times.10.sup.5 S.sup.-1 b. Substrate:
Suc-Ala-Ala-Pro-Phe-p-nitroanilide Ref 1: Toogood, H. S. Biochem.
J. 2000 250, p321-328 9. 4-phytase EC number; 3.1.3.26 from
Aspergillus fumigatus a. Turnover number: 3.5-4.1.times.10.sup.5
S.sup.-1 b. 4-nitrophenyl phosphate or myo-inositol
hexakisphophsphate Ref 1: Rodriguez, E. et al. Biochem Biophys Res
Commun. 2000 268(2) p373-378
10. Acetylcholinesterase
[0093] EC number: 3.1.1.7 a. Turnover number: 1.4.times.10.sup.5
S.sup.-1 b. Substrate: acetylcholine Ref 1: Rothenberg M. A. et al.
J. Biol. Chem. 168 (1) p223-231
11. Laccase
[0094] EC number: 1.10.3.2 from Basidiomycota a. Turnover number:
0.6.times.10.sup.5 S.sup.-1 b. substrate:
2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) Ref 1:
Jordaan, J. et. al., Enzyme Microb. Technol. 2004 V34, P635-641 12.
bacterial leucyl aminopeptidase EC number: 3.4.11.10 from Fasciola
hepatica a. Turnover number: 0.3.times.10.sup.6 S.sup.-1 b.
Substrate: L-Cys-7-amido-4-methylcoumarin Ref 1: Acosta, D. et al.
Molecular and Biochem. Parasitology 2008, V158(1) p52-64.
13. Tripeptidyl-peptidase I
[0095] EC Number: 3.4.14.9 from Dictyostelium discoideum a.
Turnover number: 0.55.times.10.sup.6 S.sup.-1 b. Substrate:
Ala-Ala-Phe-p-nitroanilide Ref 1: Krimper, R. P. Biochem and
Molecular Biology International 1999 47(6) p1079-1088 14.
coagulation factor VIIa EC number: 3.4.21.21 from homo sapiens a.
Turnover number: 0.35.times.10.sup.6 b. Substrate:
N-methylsulfonyl-D-Phe-Gly-Arg-p-nitroanilide Ref 1:
Neuenschwander, P. F. et al. Biochemistry 2002 41 p3364-3371
15. Trypsin
[0096] EC number: 3.4.21.4 from Periplaneta Americana a. Turnover
number: 0.91.times.10.sup.6 S.sup.-1 b. Substrate:
o-aminobenzoyl-AGSRGAGQ-(2,3-dinitrophenyl-ethylene diamine) Ref 1:
Marana, S. R. et al. Biochemical and Biophysical Research
Communications 2002 290 p494-497
16. Beta-fructofuranosidase
[0097] EC number:3.2.1.26 from Thermotoga neapolitana a. Turnover
number: 0.73.times.10.sup.6 S.sup.-1 b. Substrate: sucrose Ref 1:
Dipasquale, L. et al. Extremophiles 2009 13 p345-354
[0098] The sensitivity and specificity of the analyte detection
could be further improved by utilizing a temperature jump or
gradient. Some of the most stable enzymes require higher
temperature for optimized activity or as a trigger to activate the
enzymes. The reaction temperature therefore could be higher than
room temperature, at which temperature, the reaction is still
viable. When the temperature is lower than the optimized region,
the activity of the enzyme is greatly reduced. The reaction
contaminants or background molecules from the sample could be
removed or neutralized first before raising the temperature of the
optimized zone. One of the reaction contaminants could be dissolved
carbon dioxide in the solution, which is an interference to pH
variation. Raising temperature will reduce the solubility of the
carbon dioxide therefore reduce the interference. The temperature
could be raised by a physical method such as concentrated solar
power or by exothermic chemical method such as enthalphy change of
solvation of magnesium sulphate or calcium chloride, or by a
electrical method such as by heat pump. One example of the heat
pump is a Peltier device. By the same token, the reaction rate
could also be deliberately reduced to optimize the reaction by
reducing the temperature. One example of the chemical method is the
solvation of ammonium nitrate. A heat pump could be used to reduce
the temperature by switching the electrical polarity. In some
embodiments, the reaction temperature is from about 4.degree. C.,
10.degree. C., 20.degree. C., 30.degree. C., 40.degree. C.,
50.degree. C., 60.degree. C., 70.degree. C., 80.degree. C.,
90.degree. C., to about 95.degree. C., or higher while still
maintaining enzyme activity, or any temperature between the said
temperatures.
[0099] In addition to the enzymes and substrate systems shown
above, the present invention can utilize one or two or more
additional pro-enzymes (B, C, D, or more). In such embodiments,
pro-enzyme B as a substrate could be activated by an enzyme
cassette A in the reporting group. Once enzyme B is activated,
enzyme B can further activate enzyme C, which will activate enzyme
D and so on. The effective turnover constant in such linked systems
is the multiple of the turnover constant of each activated
pro-enzyme. For example, the turnover constant of each enzyme (B,
C, D, and more) could be as low as 10 or 102 per second per enzyme.
However, the multiplied turnover number would be more than 104 per
second per enzyme. An example includes the blood-clotting cascade,
where activated factor Xa activates thrombin, which activates
fibrin from fibrinogen. The activated thrombin also activates
factor Xa via factor Villa and IXa.
C. Formation of Reporter Carrier
[0100] The carrier can be associated with a proficiency enzyme or
carrier enzyme either noncovalently or covalently. Examples of
conjugation include, but are not limited to, cloning and expressing
chimeric proteins as a carrier; use of chemically modifying
carbodiimide-NHS reaction, S--Au complex, Tosyl-amine reaction,
formaldehyde conjugation, disulfide bonding, molecular imprinting,
and site-directional conjugation or random conjugation using any of
these methods; and non-convalently: through chimera biotin-avidin
or thrombin-hirudin association.
[0101] Further examples of coupling or cross linking chemistry that
may be used to link two molecules of interest in a process, often
referred to as bioconjugation, are known in the art. Common
coupling chemistries utilize amine coupling of lysine amino acid
residues (typically through amine-reactive succinimidyl esters) and
sulfhydryl coupling of cysteine residues (via a sulfhydryl-reactive
maleimide). Other linkages can be generated by cycloaddition
reactions or "click chemistry". See, .e.g., Bioconjugation
Techniques, Greg. T. Hermanson, Academic Press, 1996; "Advances in
Bioconjugation", Kalia, J. and Raines, R. T., Current Organic
Chemistry, 14: 138-147 (2010).
[0102] Alternatively, the carrier may be made as a fusion protein
using standard molecular biology and protein purification
techniques if two proteins are joined. Once sequences encoding a
desired fusion protein have been prepared, they can be cloned into
any suitable vector or replicon. Numerous cloning vectors are known
to those of skill in the art, and the selection of an appropriate
cloning vector is a matter of choice. Examples of recombinant DNA
vectors for cloning and host cells which they can transform include
the bacteriophage .lamda. (E. coli), pBR322 (E. coli), pACYC 177
(E. coli), pKT230 (gram-negative bacteria), pGV1106 (gram-negative
bacteria), pLAFR1 (gram-negative bacteria), pME290 (non-E. coli
gram-negative bacteria), pHV14 (E. coli and Bacillus subtilis),
pBD9 (Bacillus), pIJ61 (Streptomyces), pUC6 Streptomyces), YIp5
(Saccharomyces), YCp19 (Saccharomyces) and bovine papilloma virus
(mammalian cells). See, Sambrook et al., supra; DNA Cloning, supra;
B. Perbal, supra. The gene can be placed under the control of a
promoter, ribosome binding site (for bacterial expression) and,
optionally, an operator (collectively referred to herein as
"control" elements), so that the DNA sequence encoding the desired
protein is transcribed into RNA in the host cell transformed by a
vector containing this expression construction. The coding sequence
can or can not contain a signal peptide or leader sequence. Leader
sequences can be removed by the host in post-translational
processing. See, e.g., U.S. Pat. Nos. 4,431,739; 4,425,437;
4,338,397.
[0103] The expression vector is then used to transform an
appropriate host cell. A number of mammalian cell lines are known
in the art and include immortalized cell lines available from the
American Type Culture Collection (ATCC), such as, but not limited
to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster
kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular
carcinoma cells (e.g., Hep G2), Madin-Darby bovine kidney ("MDBK")
cells, as well as others. Similarly, bacterial hosts such as E.
coli, Bacillus subtilis, and Streptococcus spp., will find use with
the present expression constructs. Yeast hosts useful in the
present invention include, but are not limited to, Saccharomyces
cerevisiae, Candida albicans, Candida maltosa, Hansenula
polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia
guillerimondii, Pichia pastoris, Schizosaccharomyces pombe and
Yarrowia lipolytica. Insect cells for use with baculovirus
expression vectors include, but are not limited to, Aedes aegypti,
Autographa californica, Bombyx mori, Drosophila melanogaster,
Spodoptera frugiperda, and Trichoplusia ni.
Capture Component
[0104] A capture component can be immobilized to the surface of a
membrane for the capture of reporter carrier-target complexes. The
capture molecule recognizes and forms a complex with the target
without preventing the reporter carrier from forming a complex with
the same target molecule. Only a reporter carrier that has formed a
complex with the target will be immobilized by the capture
component. As shown in FIG. 1, immobilization of reporting
carrier-target capture component results in the formation of a
sandwich structure. The reporting carriers that are not part of the
structure are soluble and are removed by flow of liquid or by
washing, for example, in a lateral flow format. When a wash
solution is used, a buffer having a pH of about 8 to about 9.5 is
preferred. The buffering capacity of the buffer must not be greater
than the expected pH change when a when a proton producing enzyme
and substrate are used. Typical wash buffer solutions would have a
concentration of about 0.03 mM to about 0.1 mM. Aqueous, alcohol
aqueous and saline buffers comprising
tris(hydroxymethyl)aminomethane (Tris) and phosphates are
non-limiting examples of suitable buffering solutions. In general,
the capture component will be a protein or nucleic acid. The
capture component can be immobilized to the surface of a
chromatographic medium using methods known in the art. See, e.g.,
Nakanishi et al., Current Proteomics, 5:161-197 (2008). Methods for
immobilization of nucleic acids to surfaces are also known. See,
e.g., Wu et al., J. Biomater. Sci. Polymer. Edn., 19: 725-753
(2008). Multiple types of capture components and reporter carriers
may be used to detect multiple targets in one test. For example,
multiple targets can be multiple nucleic acids or a combination of
nucleic acid and non-nucleic acid, e.g., protein targets. Any of a
number of immobilization methods, both covalent and non-covalent,
known in the art may be used in the practice of the invention.
General Detection Method
[0105] FIG. 1 shows the various stages for general target
detection, involving sample preparation, reporting carrier
attachment to a target, signal carrier-target immobilization,
excess reagent wash away, and result display.
[0106] Prepare sample: As a first step, sample is prepared for use
in the method and device of the present invention. As will be
appreciated by one of skill in the art, possible steps within this
stage will vary with implementation and target molecule types.
Examples of steps that may be used in this stage include, but are
not limited to: methods to improve concentration of target
molecules, nucleic acid extraction from cells, and purification of
sample. In some embodiments, extensively sample preparation is not
needed prior to application of the sample (e.g., a liquid sample
like blood or urine).
[0107] Attach reporting carrier: As shown in FIG. 1, this step uses
reporting carrier containing enzyme cassettes. The reporting
carrier will form a complex with target molecules of interest.
[0108] Immobilize reporting carrier: an immobilizer array of
capture components is used to capture the biological targets. As
shown in FIG. 1, upon immobilizer capture of a target which is in a
complex with a reporting carrier, a sandwich structure of
immobilizer-biological target reporting carrier is formed. In
different embodiments, it is possible to have multiple immobilizers
(or capture components) in the array for different target
molecules.
[0109] Wash away excess molecules: This optional step washes away
other molecules, excess sample, reporting carriers, or any other
molecules which are not captured by the immobilizer arrays. A wash
solution is applied upstream of the detection zone such that it
migrates by capillary action through the detection zone and the
control zone if present to the absorptive pad at the distal end of
the test strip. The amount of the optional wash solution depends on
the size of the test strip and the nature of the test and is easily
determined by one skilled in the technology.
[0110] Display result: The immobilized reporting carrier at this
stage is mixed with reagents, generally substrates, which react
with a proficient or high yield enzyme in the carrier to rapidly
form products which can be observed. One or more enzymes could also
be added to accelerate the formation of the products thus foaming a
reaction cascade. Depending on the selection of the enzymes and
reagents, different detection methods can be employed. These
include, but are not limited to, for example pH change, color
change, density of color change, or light emissions, among others.
Substrates are typically applied to the chromatographic test strip
by spray, painting, dropper or poured.
[0111] FIG. 2 illustrates the application of the present invention
in a nucleic acid test. The general workflow is similar to that
shown in FIG. 1, as applied to nucleic acid target molecules.
[0112] Prepare sample: For this application, sample preparation
could include routine steps such as nucleic acid extraction, sample
concentration, RNA extraction, and nuclease inhibition. For some
applications, the DNA, e.g., genomic DNA may need to be broken down
in size, for instance, by shearing before use in the method.
[0113] Attach reporting carrier: In this application, the high
proficiency or high yield enzymes are attached to synthesized
polynucleotides which form the reporter carrier or enzyme cassette.
In some embodiments, there can be more than one polynucleotide for
a nucleic acid target. The polynucleotides in the reporter carrier
are allowed to hybridize to the target nucleic acid.
[0114] Immobilize reporting carrier: As shown in FIG. 2, the
immobilizer arrays can comprise one or more polynucleotides which
are specific to the target nucleic acid. If there is a match, the
immobilizer will hybridize with the target nucleic acid to form a
complex of reporting carrier-target-immobilizer. The formation of
this complex locks the reporting carriers in place. The selection
of polynucleotides in reporting carriers and immobilizers can be
used for a variety of applications, including but not limited to,
detection of single nucleotide variation, methylation, or
sequencing of a target.
[0115] Wash away excess molecules: At this stage, the excess
molecules and enzyme cassettes are washed away, leaving behind the
bound reporting carrier-target complex.
[0116] Display signal: The reporting carrier would then be allowed
react with substrate reagents to form an observable product. In
some embodiments, an additional one or more enzymes could be added
to accelerate the reaction.
Lateral Flow Platform
[0117] FIG. 3 illustrates implementation of the present invention
in a lateral flow platform. It will be appreciated that
implementation may vary for different sample types with respect to
the location of different components at various sections of the
platform for optimal performance. In FIG. 3, The sample pad 1 is
upstream of conjugate pad 2 which is upstream of Chromatographic
membrane 3 which is upstream of detection zone (Test Line) 4, which
is upstream of Control zone (control line) 5, which is upstream of
Adsorbent pad 6. The entire devise is on support material 8.
[0118] Similar to other forms of lateral flow, the sample can enter
the device at a sample pad, and ultimately arrive at the absorbent
pad by traveling along the membrane on top of the backing material.
The reporting carriers can be deposited in a conjugate pad, and the
immobilizer array can be present in sense and control lines. As is
easily appreciated, the order of the control and the sense lines
can be different, and there can be multiple sense lines in some
embodiments. The substrate reagent and additional enzymes (if
needed) can also be present in the control and sense lines.
[0119] As the sample travels through the conjugate pad, a complex
of reporting carrier and the target are formed. When the sample
front reaches the sense line, the target, which has formed a
complex with the reporting carrier, would be immobilized by the
array. Reaction then occurs to form sufficient products that can be
monitored. If the immobilized probe (capture component) does not
match with a target in the sample, then the sample nucleic acid
would flow through the membrane leaving minimal concentration of
enzyme at the detection line. A control line acts as the positive
control on the device. The positive control demonstrates that the
test strip is functional, thereby minimizing false negative test
results.
[0120] FIG. 7 illustrates a positive test result. The detection
zone 4 indicates the presence of enzyme indicating that the
analyte-reporting carrier complex has been captured in the
detection zone. The positive indicator response is the result of
substrate being added to the test strip after migration of the test
sample past the detection zone. The control zone 5 also indicates
the presence of enzyme indicating that the uncomplexed reporting
carrier has migrated the length of the test device and has been
captured in the control zone providing a change in the indicator
after application of the enzyme substrate establishing the presence
of the uncomplexed reporting carrier.
[0121] FIG. 8 illustrates a negative test result as none of the
analyte reporting carrier complex was formed or captured in the
detection zone. The positive response indicated in the control zone
verifies that the test strip functioned properly and that the
exzyme and reporting carrier are present and active.
[0122] The following examples of specific aspects for carrying out
the present invention are offered for illustrative purposes only,
and are not intended to limit the scope of the present invention in
any way.
EXAMPLES
Example 1
Embodiments of a Lateral Flow Device
[0123] FIG. 4 shows various embodiments of a lateral flow device
that can be used in the practice of the present invention. A sample
loading zone 1 is provided. Flow in the direction indicated by the
arrow results in the interaction of the sample with a reporting
carrier 7 found in the reporter carrier loading zone 2. The
detection zone 4 contains capture components (upwardly directed
solid arrows) and display result materials, such as substrates,
and, if necessary, additional pro-enzymes to further amplify the
signal. A positive control zone can be provided 5. The supporting
scaffold matrix through which the sample moves, if present, can be
a chemical coating or membrane of porous matrix or branched
polymers. As shown in FIG. 4B-4D, the scaffold matrix may be
mounted on a solid non-porous backing 8, which may be flexible or
porous. In some embodiments, the detection zone 1, can contain one
type of immobilizer array 8 (such as a antibody, antigen, nucleic
acid, etc.) or multiple types of immobilizer array, which may be
placed at different spatial locations in the detection zone 4a, 4b,
and 4c. FIG. 4E illustrates that inactive pro-enzymes 9, can be
present in the immobilizer array, which when activated can lead to
a high overall turnover for the system. In further embodiments, the
supporting scaffold matrix in the vicinity of the immobilizer array
may comprise a pH sensitive material, such as a pH sensitive
hydrogel containing 10, for example gold nanoparticles 10a. Such a
pH sensitive material may be used to detect the reaction of an
enzyme, whose catalytic activity results in a pH change. As shown
in FIGS. 4G and 4H, examples of surface pH sensitive display
molecules that can be used to practice the present invention
include acridine orange, cyanine, liposomes, or fluorescent dextran
11 or a soluble molecule as such as BSA 12.
Example 2
Operation of Embodiments of a Lateral Flow Device
[0124] FIG. 5 illustrates operation of various embodiments of the
present invention. A sample target 13 can be introduced into a
sample loading zone 1 (Step A). Lateral flow in the direction
indicated by the arrow can bring the sample target 13 into contact
with a reporting carrier 7 (Step B). Further lateral flow carries
the complex of sample target and reporting carrier 14 toward a
detection zone 5 (Step C). Capture components in the detection zone
(upwardly directed solid arrows) immobilize the complex of sample
target and reporting carrier 15 (Step D and 4-E). In various
embodiments, the capture components may be singular or clusters of
variants (compare upwardly directed arrows in Steps D with E).
Lateral flow along the capture component and immobilized complex of
sample target and reporting carrier removes excess materials such
as unbound reporting carriers, unbound sample targets and the like,
and serves as a washing step (Step F). In Step G, enzyme substrate
and other soluble molecules can be applied to the immobilizer array
zone. In various embodiments, the enzyme can be a single proficient
enzyme that converts a substrate to product 17, 18, or multiple
linked pro-enzymes 19 can be used. A number of possible display
options are shown in Steps J-L. For example, detection of reaction
product 17 can be indicated through the change of color of a
hydrogel, such as a pH sensitive hydrogel containing gold
nanoparticles 10, 10b, 21, 22 (Step J). Other display options can
rely on a color change (e.g., silver reduction) 11, 23, 24 (Step
K). In another embodiment, the pH sensitive precipitation of a
soluble molecule such as BSA can be used for visualization 12, 25
(Step L).
[0125] In a preferred embodiment of the current disclosure, the
device is either a lateral flow unit. The whole device is very low
cost, portable, instrument-free, and sensitive. The device is
characterized by distinct aspects. One is that the enzyme is
attached to the conjugate and the other is that the indicator is
printed or immobilized at the detection zones. The product from the
enzymatic reaction reacts with the indicator to give visual
difference at the detection zones.
[0126] The indicator could be pH sensitive which will change color
or fluorescence when the protonation status of the indicator
changed. In one of the embodiments, the pH indicator has been
reacted with a carrier membrane molecule such as cellulose acetate.
In another embodiment, the pH indicator is encapsulated in proton
permeable plastics ["Full-range optical pH sensor based on imaging
techniques", Capel-Cuevas, S., Cuellar, M. P., de Orbe-Paya, I.,
Pegalajar, M. C., Capitan-Vallvey, L. F., Analytica Chimica Acta,
681 (2010) 71-81. In both cases, the deposition of the pH indicator
onto the membrane did not interfere with the conjugate binding in a
lateral flow device.
[0127] In a preferred embodiment, the indicator is printed or
sprayed on to a membrane down stream of the conjugate pad to form a
line or a zone. Each line or zone is separated from another line or
zone of indicator by a buffering region of membrane without the
indictor. The buffering region could also contain a layer of
immobilized buffer component to prevent the cross reaction between
different lines/zones.
[0128] In one embodiment, the dye (bromothymol blue) is prepared
mixing bromothymol blue 2.1 mg; tridodecylmethylammonium chloride
(TDMAC) 2.8 mg; dioctyl sebacate (DOS) 19.6 mg; ethylenglycol 19.6
mg and tetrhydrofuran (THF) 1 ml. The bromothymol solution has a pH
of about 8 to about 9.5. The indicator-polymer solution was spray
painted on the membrane using an air brush gun. The airbrush gun is
the High Performance C PLUS Airbrush made by Iwata. Only the
regions of the control/testing line on the lateral flow strip were
coated with the polymer/indicator. After spraying, the whole strip
was left at desiccator for 16 hrs before use.
[0129] In a preferred embodiment, a pH indicator could be
covalently linked with polymers such as cellulose (U.S. Pat. No.
4,029,597). In one embodiment, the pH sensitive dye is specially
designed with hydroxysulfonyl end group for covalent chemistry of
the dye molecule and cellulose. PH sensitive dye molecule is
treated with sulphuric acid at room temperature to form terminal
sulfoester groups. Later, the dye solution in sulphuric acid is
diluted into de-ionized water and neutralized with sodium
hydroxide. Cellulose acetate is added to the solution at this
stage. After 5 min, sodium carbonate and in another 5 min sodium
hydroxide is added to the solution. The sulfoester group under the
strong alkaline conditions form vinyl sulfone end group and
cellulose acetate is hydrolysed to form cellulose with terminal
hydroxyl groups. Under the same alkaline conditions, vinyl sulfone
group on the dye molecule reacts with the hydroxyl groups of
cellulose to form a covalent linkage between pH indicator dye and
cellulose. The final reaction product is washed with de-ionized
water. The pH sensitive cellulose is dissolved in solvent to
prepare 10% wt solution. Aqueous solution is preferred. Indicator
solution is spray coated on the nitrocellulose membrane to have pH
indicator dye immobilization on the lateral flow device.
[0130] A preferred substrate solution for the carboxylesterase
enzyme comprises the following formula:
10 mM allyl hexanoate
0.1 mM Tris pH 8.5
[0131] 5% isopropanol
[0132] The substrate is the allyl hexanoate. Upon hydrolysis, one
allyl hexanoate molecule produces one hexanoic acid and one
2-propenol. The starting pH of the reaction is defined by the 0.1
mM Tris at pH 8.5. The buffer capacity of the substrate solution
would be less than 50 micro molar. The overall proton balance at pH
8.5 would produce proton surplus, such that the pH value would be
reduced as the result of the hydrolysis.
[0133] The change in pH as a result of hydrolysis will cause the
indicator of for example bromothymol blue as described above to
change color from blue to yellow.
[0134] In a preferred embodiment, the device is protected from the
carbon dioxide in the air, which would gradually move solution pH
toward pH 6.5. The protection could be done by lamination or
housing.
[0135] Preferred carriers are micro particles having diameters from
about 10 nm to about 500 nm. Preferably the particles will have
diameters from about 10 nm to about 100 nm. Preferred particles are
of latex or gold. The particles are coated with material the
proficient enzyme can bond or complex with. A preferred coating is
streptavidin. A preferred particle is a 20 nm gold particles with
streptavidin coating. A preferred enzyme is carboxylesterase.
Preferred substrates for carboxylesterase are phenethyl butyrate in
0.1 mM Tris buffer at pH 8 and 10 mM allyl hexanoate in 0.1 mM Tris
pH 8.5 buffer in 5% isopropanol.
[0136] A preferred particle composition is a 40 nm Innovacoat gold
particle coated with Streptavidin by the following protocol:
1. Prepare 0.2 mg/mL streptavidin solution in phosphate buffer, pH
7.4 2. Mix 42 .mu.L of the reaction buffer from the Innovacoat Gold
kit
<http://www.inovabiosciences.com/gold-conjugation-kits/innovacoat-gold-
.html> (20 OD 40 nm), with 12 .mu.L of the streptavidin solution
3. Transfer 45 .mu.L of the mixture to one portion of the
Innovacoat gold. 4. Incubate at room temperature for 10 mins before
stopped by 5 .mu.L of the quencher in the kit. 5. Spin down the
particle and remove the supernatant 6. Resuspend with 100 .mu.L PBS
buffer 7. Repeat step 5 8. Resuspend in 50 .mu.L PBS and add to
final 0.1% BSA and 0.01% (20K MW) polyethylene glycol 9. Mix 1
.mu.L of biotinylated antibody(about 12 .mu.g/mL), 2 .mu.L of
biotinylated carboxylesterase (about 7 ug/ml), 2 .mu.L of the
Streptavidin modified Innovacoat gold to form the reporting carrier
i.e. conjugate.
[0137] While specific aspects of the invention have been described
and illustrated, such aspects should be considered illustrative of
the invention only and not as limiting the invention as construed
in accordance with the accompanying claims.
[0138] All publications and patent applications cited in this
specification are herein incorporated by reference in their
entirety for all purposes as if each individual publication or
patent application were specifically and individually indicated to
be incorporated by reference for all purposes.
[0139] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be readily apparent to one of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications can be made thereto without
departing from the spirit or scope of the appended claims.
* * * * *
References