U.S. patent application number 10/704470 was filed with the patent office on 2004-05-20 for methods for preparing an electrosensor having a capture reagent.
Invention is credited to Zhang, Honghua.
Application Number | 20040096991 10/704470 |
Document ID | / |
Family ID | 22607271 |
Filed Date | 2004-05-20 |
United States Patent
Application |
20040096991 |
Kind Code |
A1 |
Zhang, Honghua |
May 20, 2004 |
Methods for preparing an electrosensor having a capture reagent
Abstract
The present invention relates to devices comprising
electrosensors containing capture reagents, their preparation
thereof, and their use for detecting, preferably, quantitative
measurement, of analyte in a liquid sample. In particular, the
invention relates to an enzyme electrosensor, e.g.,
electroimmunosensor, device for electrochemical detection and
preferably, real-time measurement, which is suitable for use at
point-of-care settings by unskilled personnel.
Inventors: |
Zhang, Honghua; (San Diego,
CA) |
Correspondence
Address: |
Peng Chen
Morrison & Foerster LLP
Suite 500
3811 Valley Centre Drive
San Diego
CA
92130-2332
US
|
Family ID: |
22607271 |
Appl. No.: |
10/704470 |
Filed: |
November 7, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10704470 |
Nov 7, 2003 |
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09699140 |
Oct 27, 2000 |
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6670115 |
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60167409 |
Nov 24, 1999 |
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Current U.S.
Class: |
436/518 ;
435/6.11; 436/525 |
Current CPC
Class: |
G01N 33/558 20130101;
Y10S 435/97 20130101; Y10S 435/975 20130101; G01N 33/5438
20130101 |
Class at
Publication: |
436/518 ;
436/525; 435/006 |
International
Class: |
G01N 033/543 |
Claims
What is claimed is:
1. A device for detecting an analyte in a liquid sample, which
device comprises: a) a solid support; b) an electrosensor
immobilized on said solid support, said electrosensor comprises a
working electrode and another electrode used as auxiliary and/or
reference electrode; c) a capture reagent immobilized on said
working electrode, said capture reagent is capable of binding to an
analyte; and d) conductive leads for connecting said electrodes to
a readout device for electrochemical measurement.
2. The device of claim 1, wherein the solid support comprises a
material selected from the group consisting of plastic, polyvinyl
chloride (PVC), polyvinylidene fluoride (PVDF), paper, nylon,
fiberglass, polyethylene, nitrocellulose, a wicking member having
an open mesh structure and a combination thereof.
3. The device of claim 1, wherein the electrosensor comprises a
working electrode, an auxiliary electrode and a reference
electrode.
4. The device of claim 3, wherein the working electrode and/or
auxiliary electrode comprise(s) a screen-printed carbon conductor
and the reference electrode comprises a screen-printed silver or
silver/silver chloride conductor.
5. The device of claim 1, wherein the capture reagent is selected
from the group consisting of a cell, a cellular organelle, an
inorganic molecule, an organic molecule and a mixture thereof.
6. The device of claim 5, wherein the organic molecule is selected
from the group consisting of an amino acid, a peptide, a protein, a
nucleoside, a nucleotide, an oligonucleotide, a nucleic acid, a
vitamin, a monosaccharide, an oligosaccharide, a carbohydrate, a
lipid and a complex thereof.
7. The device of claim 6, wherein the protein is an antibody,
avidin/strepavidin, protein A or protein G.
8. The device of claim 1, wherein the capture reagent is
immobilized on the working electrode via a buffered organic
solution containing a ketone or an aliphatic alcohol.
9. The device of claim 8, wherein the aliphatic alcohol is
isopropyl alcohol.
10. The device of claim 1, wherein the capture reagent is capable
of specifically binding to the analyte.
11. The device of claim 1, wherein the analyte is selected from the
group consisting of a cell, a cellular organelle, an inorganic
molecule, an organic molecule and a mixture thereof.
12. The device of claim 1, wherein the analyte is
alpha-fetoprotein, prostate-specific antigen, cardiac troponins,
c-reactive protein (CRP), or human chorionic gonadotropin, or a
marker for HBV, HAV, HCV or HIV infection.
13. The device of claim 1, further comprising an analyte bound to
the capture reagent, said analyte contains a label that is capable
of generating electrocurrent under suitable conditions.
14. The device of claim 13, wherein the label is an enzyme.
15. The device of claim 14, wherein the enzyme is horseradish
peroxidase and the enzymatic substrate is hydrogen peroxide and the
electron transfer mediator is ferrocene, or a derivative thereof,
benzoquinone, ascorbic acid or 3,3',5,5' tetramethylbenzidine.
16. The device of claim 1, further comprising a cover casing having
a liquid sample application aperture and a detection aperture.
17. The device of claim 1, further comprising a sample application
area that is separate, but in fluid communication with the
electrosensor.
18. The device of claim 17, wherein the sample application area
contains deposited labeled analyte, said labeled analyte is capable
of being dissolved or suspended into the sample liquid and being
carried to the capture reagent immobilized on the electrosensor by
the sample fluid, and said label is capable of generating
electrocurrent under suitable conditions.
19. The device of claim 17, wherein the sample application area
contains deposited labeled detection reagent, said labeled
detection reagent is capable of being dissolved or suspended into
the sample liquid, binding to the analyte, if there is any, being
carried to the capture reagent immobilized on the electrosensor by
the sample fluid to form a sandwich comprising the immobilized
capture reagent-analyte-labeled detection reagent, and said label
is capable of generating current under suitable conditions.
20. The device of claim 19, wherein the movably bound labeled
detection reagent is capable of specifically binding to the
analyte, if there is any, in the sample fluid.
21. The device of claim 19, wherein the movably bound labeled
detection reagent is an antibody.
22. The device of claim 19, wherein the label is an enzyme.
23. The device of claim 22, wherein the enzyme is horseradish
peroxidase and the enzymatic substrate is hydrogen peroxide and the
electron transfer mediator is ferrocene, or a derivative thereof,
benzoquinone, ascorbic acid or 3,3',5,5' tetramethylbenzidine.
24. The device of claim 17, wherein the sample application area is
in fluid communication with the electrosensor via a wicking
member.
25. The device of claim 24, wherein the wicking member comprises
nylon, cellulose or paper.
26. The device of claim 24, wherein the wicking member comprises a
nylon mesh having mesh opening in the range from about 0.45 .mu.m
to about 100 .mu.m.
27. The device of claim 24, wherein the wicking member provides for
substantially two dimensional transport of fluids from the
application area to the electrosensor.
28. The device of claim 17, further comprising a filter in the
application area, said filter is capable of removing insoluble or
insuspendable material(s) from the sample fluid.
29. The device of claim 28, wherein the filter is adapted for
removing insoluble or insuspendable material(s) from a sample
blood.
30. The device of claim 1, further comprising an absorptive sink in
fluid communication with the electrosensor, said sink having
sufficient porosity and capacity to absorb excess liquid or allow
excess liquid to be washed out of the device.
31. The device of claim 30, wherein the absorptive sink is a pad of
absorbent material.
32. The device of claim 1, which comprises an absorptive sink, an
electrosensor and an application area that are linearly arranged in
order.
33. The device of claim 1, further comprising an enzyme substrate
and an electron transfer mediator localized on or in proximity to
the electrosensor, said substrate and mediator can be controllably
released.
34. A device for detecting an analyte in a liquid sample, which
device comprises: a) a base sensor strip having a working
electrode, a reference electrode, and an auxiliary electrode coated
on a plastic substrate, whereon a capture reagent is immobilized on
the working electrode, said sensor strip having conductive leads
for attaching the electrodes to a readout device for
electrochemical measurement; b) a cover casing having a liquid
sample application aperture and a detection aperture; c) an
application zone for receiving a fluid containing an analyte from
the application aperture, said application zone, in the dry unused
form, containing a labeled detection reagent capable of
specifically binding to said analyte, wherein the said labeled
reagent is released into mobile form when in contact with the
liquid sample; d) a detection zone in fluid communication with the
electrodes in the presence of a liquid sample received from the
detection aperture; e) a wicking member that carries the liquid
sample from the application zone to the detection zone by capillary
action, wherein said analyte is sandwiched between the detection
reagent and the capture reagent immobilized on the electrode
surface; and f) an absorbent sink placed in partial contact with
the wicking member at the end of the flow path to absorb any excess
fluid from the detection zone.
35. A method for assaying an analyte in a liquid sample, which
method comprises: a) contacting a liquid sample containing or
suspected of containing an analyte with the device of claim 1 under
suitable conditions whereby the analyte, if there is any, binds to
the capture reagent immobilized on the working electrode and the
binding between the analyte and the capture reagent causes a change
in the current that is capable of being detected by the
electrosensor of the device; and b) detecting the change in the
electrocurrent generated in step a), whereby the presence or amount
of the analyte in the sample is assessed.
36. The method of claim 35, wherein the capture reagent is an
antibody.
37. The method of claim 35, wherein the capture reagent is capable
of specifically binding to the analyte.
38. The method of claim 35, wherein the device further comprises an
analyte bound to the capture reagent, said analyte contains a label
that is capable of generating electrocurrent under suitable
conditions, and the binding between the unlabeled analyte in the
sample and the capture reagent displaces the labeled analyte from
the capture reagent and decreases electrocurrent that is capable of
being detected by the electrosensor of the device.
39. The method of claim 35, wherein the device further comprises a
sample application area containing deposited labeled analyte, said
labeled analyte is capable of being dissolved or suspended into the
sample liquid and being carried to the capture reagent immobilized
on the electrosensor by the sample fluid, said label is capable of
generating electrocurrent under suitable conditions, and the
presence of unlabeled analyte in the sample fluid decreases
electrocurrent that is capable of being detected by the
electrosensor of the device.
40. The method of claim 35, wherein labeled analyte is added in the
sample fluid or added separately, said labeled analyte is capable
of being dissolved or suspended into the sample liquid and being
carried to the capture reagent immobilized on the electrosensor by
the sample fluid, said label is capable of generating current under
suitable conditions, and the presence of unlabeled analyte in the
sample fluid decreases current that is capable of being detected by
the electrosensor of the device.
41. The method of claim 35, wherein the device further comprises a
sample application area containing deposited labeled detection
reagent, said labeled detection reagent is capable of being
dissolved or suspended into the sample liquid, binding to the
analyte, if there is any, being carried to the capture reagent
immobilized on the electrosensor by the sample fluid to form a
sandwich comprising the immobilized capture reagent-analyte-labeled
detection reagent, and said label is capable of generating
electrocurrent under suitable conditions.
42. The method of claim 41, wherein the deposited labeled detection
reagent is capable of specifically binding to the analyte, if there
is any, in the sample fluid.
43. The method of claim 41, wherein the deposited labeled detection
reagent is an antibody.
44. The method of claim 35, wherein a labeled detection reagent is
added in the sample fluid or added separately, said labeled
detection reagent is capable of being dissolved or suspended into
the sample liquid, binding to the analyte, if there is any, being
carried to the capture reagent immobilized on the electrosensor by
the sample fluid to form a sandwich comprising the immobilized
capture reagent-analyte-labeled detection reagent, and said label
is capable of generating electrocurrent under suitable
conditions.
45. The method of claim 44, wherein the movably bound labeled
detection reagent is capable of specifically binding to the
analyte, if there is any, in the sample fluid.
46. The method of claim 44, wherein the deposited labeled detection
reagent is an antibody.
47. The method of claim 35, wherein the sample application area is
in fluid communication with the electrosensor via a wicking
member.
48. The method of claim 35, wherein the device further comprises a
filter in the application area, said filter is capable of removing
insoluble or insuspendable material(s) from the sample fluid.
49. The method of claim 35, wherein the device further comprises an
absorptive sink in fluid communication with the electrosensor, said
sink having sufficient porosity and capacity to absorb excess
liquid or allow excess liquid to be washed out of the device.
50. The method of claim 35, wherein the device comprises an
absorptive sink, an electrosensor and an application area that are
linearly arranged in order.
51. The method of claim 35, wherein the device further comprises an
enzyme substrate and an electron transfer mediator localized on or
in proximity to the electrosensor, said substrate and mediator are
controllably released to generate current that is capable of being
detected by the electrosensor of the device.
52. The method of claim 35, wherein an enzyme substrate and an
electron transfer mediator are added in the sample fluid or added
separately for generating electrocurrent that is capable of being
detected by the electrosensor of the device.
53. The method of claim 35, wherein the analyte is a marker for a
biological pathway, a stage of cell cycle, a cell type, a tissue
type, an organ type, a developmental stage, a disease, disorder or
infection type or stage, or drug or other treatments.
54. The method of claim 35, wherein the analyte is
alpha-fetoprotein, prostate-specific antigen, cardiac troponins,
c-reactive protein (CRP), or human chorionic gonadotropin or a
marker for HBV, HAV, HCV or HIV infection.
55. The method of claim 35, wherein the liquid sample is buffer,
blood, serum, plasma, or urine.
56. A method for assaying an analyte in a liquid sample, which
method comprises: a) contacting a sample with a solution containing
a labeled detection reagent that specifically binds to an analyte
in the sample to form an assay mixture; b) incubating the assay
mixture with a device of claim 1 for a time period sufficient for
the analyte to become sandwiched between the labeled detection
reagent and the capture reagent immobilized on the surface of the
sensor; c) rinsing the electrosensor with an appropriate buffer
solution; d) adding a detection solution containing a substrate and
an electron transfer mediator to the sensor surface to initiate an
electron transfer reaction; and e) determining current response
generated from the electron transfer mediator catalyzed by the
labeled detection reagent, whereby the presence or amount of
analyte in the liquid sample is assessed.
57. A method for assaying an analyte in a liquid sample, which
method comprises: a) applying a fluid sample containing the analyte
of interest to the application zone of the device of claim 34, b)
allowing the liquid sample to transport from application zone to
the detection zone by capillary action, wherein the analyte is
sandwiched between the labeled reagent and the capture reagent
immobilized on the sensor surface; c) adding a detection solution
containing a substrate and an electron transfer mediator through
the detection aperture to the detection zone to initiate an
electron transfer reaction; and d) amperometrically determining
current response generated from the electron transfer mediator
catalyzed by the labeled detection reagent, whereby the presence or
amount of the analyte in the liquid sample is assessed.
58. A method for preparing an electrochemical sensor for the
detection of an analyte in a liquid sample, which method comprises
immobilizing a capture reagent capable of binding to an analyte on
the surface of a hydrophobic, non-metal electrode by contacting
said electrode surface with a solution containing said capture
reagent and an organic immobilizing agent that wets said electrode
surface and facilitates immobilization of said capture regent on
said electrode surface.
59. The method of claim 58, wherein the organic immobilizing agent
is a buffered aliphatic alcohol solution.
60. The method of claim 58, wherein the aliphatic alcohol is
isopropyl alcohol.
61. The method of claim 58, wherein the electrode is fabricated by
screen printing carbon composition upon a plastic substrate.
62. The method of claim 58, wherein the electrode is a working
electrode and is coupled with at feast one additional electrode
fabricated by screen printing a conductive composition upon a
plastic substrate.
63. The method of claim 62, wherein the working electrode and the
additional electrode are fabricated by screen printing carbon
composition upon the same plastic substrate.
64. The method of claim 62, wherein the capture reagent is selected
from the group consisting of an amino acid, a peptide, a protein, a
nucleoside, a nucleotide, an oligonucleotide, a nucleic acid, a
vitamin, a monosaccharide, an oligosaccharide, a carbohydrate, a
lipid and a complex thereof.
65. The method of claim 64, wherein the protein is an antibody,
avidin/strepavidin, protein A or protein G.
66. The method of claim 58, wherein the capture reagent is capable
of specifically binding to an analyte.
67. The method of claim 58, further comprising coating the
electrode surface containing the immobilized capture reagent with a
stabilizing solution that stabilizes the immobilized capture
reagent.
68. The method of claim 67, wherein the stabilizing solution
stabilizes the capture reagent immobilized on the electrode in a
dry form.
69. The method of claim 67 wherein the stabilizing solution
contains a sugar, a polyhydroxy compound, or StabilCoat.RTM..
70. A kit for detecting an analyte in a liquid sample, which kit
comprises: a) the device of claim 1; and b) an effective amount of
a suitable electron transfer mediator and substrate, and any other
buffer solutions, conjugate solutions or, standards necessary for
performing the detection assay.
71. A device for detecting an analyte in a liquid sample, which
device comprises a sample application area that is in fluid
communication with an electrosensor via a wicking member, wherein
the wicking member has an open mesh structure.
72. The device of claim 71, wherein the wicking member comprises a
nylon mesh having mesh opening in the range from about 0.45.mu.m to
about 100 .mu.m.
73. The device of claim 71, wherein the wicking member provides for
substantially two dimensional transport of fluids from the
application area to the electrosensor.
74. The device of claim 71, wherein a capture reagent capable of
binding to an analyte is immobilized on the electrosensor.
Description
[0001] The present application claims priority benefit of the
provisional U.S. patent application Ser. No. 60/167,409, filed Nov.
24, 1999, the content of which is herein incorporated by reference
in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to devices comprising
electrosensors containing capture reagents, their preparation
thereof, and their use for detecting, preferably, quantitative
measurement, of analyte in a liquid sample. In particular, the
invention relates to an enzyme electrosensor, e.g.,
electroimmunosensor, device for electrochemical detection and
preferably, real-time measurement, which is suitable for use at
point-of-care settings by unskilled personnel.
BACKGROUND OF THE INVENTION
[0003] There is an increasing public awareness of the need for
diagnostics to determine levels of various components in human
fluids, such as blood or serum. Of particular interest are tests
designed for non-expert use that produce rapid and quantitative
results.
[0004] Immunoassays have been widely used for the detection of
antigens and antibodies. The most commonly used immunoassays are
enzyme immunoassays (EIAs). The importance of EIAs, particularly in
clinical analyses, medical diagnostics, pharmaceutical analyses,
environmental control, food quality control, and bioprocess
analyses, lies in their high sensitivity and specificity, which
allow the detection of a wide spectrum of analytes in various
sample matrices.
[0005] EIAs are commonly referred to as either heterogeneous
(necessitating free antigen separation from those that have been
bound to antibody) or homogeneous (requiring no separation or
washing steps during the assay). Also, EIAs can be either
competitive or non-competitive, depending on the availability of
antibody binding sites. Conventional EIAs are convenient for
analysis of great numbers of samples on a routine basis and are
widely used in a broad spectrum of applications. However, these
methods require multiple washing and incubation steps to implement,
and can be utilized in high volume only by complex and expensive
analytical equipment. The need for multiple washing and incubation
steps has also limited the development of portable point-of-care
analytical devices that can be used to perform assays in
decentralized locations.
[0006] In recent years, efforts have been made to overcome the
limitations of heterogeneous EIAs and to search for homogeneous,
rapid, and separation-free immunoassays that can be readily
conducted at the point of care. Fast and simple EIA tests capable
of detecting a single analyte with a color change that can be
visually interpreted have been developed. Based on the techniques
of immobilizing antigen or antibody on a solid-phase support, assay
formats such as dipsticks, test tubes, and wicking membrane test
cartridges have been used to provide fast results for analytical
conditions where a simple qualitative (yes/no) answer is clinically
relevant. These membrane-based assays have gained increasing
popularity in many areas of clinical chemistry. They not only form
the basis of the majority of home use tests, but also are rapidly
gaining use in the physician's office and hospital lab. These tests
are widely accepted and increasingly used for detection of
pregnancy, strep throat, arid bacteria, as well as for prediction
of ovulation. Examples of such assays are described in U.S. Pat.
Nos. 5,622,871, 4703,017, 5,468,647, 5,622,871, and 5,798,273.
However, most of these rapid tests are incapable of performing
sensitive and quantitative detection. As a result, medical
diagnoses that require quantitative measurement of the target
analyte remain within the domain of the complex immunoassay
analyzers in the centralized laboratory.
[0007] A major trend in the development of rapid immunoassays is
the move toward quantitative testing. The use of membrane-based
immunoassays has been proposed for quantitative measurement of
analytes. As a specific example, U.S. Pat. No. 5,753,517 describes
a quantitative immuno-chromatographic assay utilizing
antibody-coated particles, independent control particles, and
capillary flow through a membrane. However, there are difficulties
in developing such quantitative immunoassays based on membrane
format for point-of-care diagnostic tests. Perhaps the most
significant problems with the use of membrane-based immunoassays
arise from requirements for the membrane that are contradictory.
For example, immobilization of protein in the detection area
requires that the membrane have a strong binding affinity for the
protein, but transport of analyte and particles containing
detection components demands that the membrane not bind to protein.
Furthermore, factors commonly used for increasing the performance
of the membrane assay are often mutually exclusive. For example,
blocking reagents that reduce nonspecific interactions usually also
reduce the amount of specific signal. In light of these competing
requirements commonly seen in efforts to develop membrane-based
immunoassays, it becomes clear that conventional membrane systems
have limited advantages for use in quantitative immunoassays.
[0008] Accordingly, there is a need to develop improved assays,
e.g., immunoassays, that can provide rapid, quantitative, and
reliable results. The high sensitivity of electrochemical detection
coupled with the inherent specificity of antibody-antigen reactions
has resulted in a remarkable technique known as electrochemical
immunoassay. The advantages of such assays include, among others,
the ability to measure untreated samples in the presence of
possible interfering substances, as well as the simplicity, and
sensitivity associated with electrochemical detection.
[0009] Immunoassays employing amperometric electrochemical
detection have been applied to the determination of analytes in
fluid samples. An immunoassay device using amperometric detection
to perform diagnostic tests for analytes in body fluids is
described, as a specific example, in U.S. Pat. No. 5,830,680. The
device includes an electrochemical detection system for a
separation-free sandwich-type immunoassay, in which a protein
analyte such as human chorionic gonadotropin (hCG) is sandwiched
between a capture antibody immobilized on a microporous membrane
gold electrode and an alkaline phosphatase-labeled antibody.
Although such a device offers a separation-free feature, the time
required for manipulating and incubating the sample limit the use
of such assays for rapid diagnostic testing.
[0010] A method employing liposomes for signal production and
electrochemical detection in immunoassays is described in U.S. Pat.
No. 5,756,362. In these assays, liposomes that encapsulate an
electroactive marker are conjugated with an analyte. A test device
first allows incubation of a sample containing an analyte with a
binding material specific for the analyte and the analyte-liposome
conjugate. Following incubation, the mixture solution is allowed to
traverse through an absorbent material strip to reach an
electrochemical measurement portion where the liposome is lysed by
a lysing reagent to release the electroactive marker. The amount of
marker released is then detected electrochemically and correlated
with the amount of analyte in the sample.
[0011] In the methods described in U.S. Pat. No. 5,391,272,
bioactive components are coated onto colloidal gold and
subsequently coated onto a sensor. Detection of analyte is achieved
by measuring current generated by an electroactive species bound to
the sensor as part of an analyte/enzyme catalytic response.
Although the method is suitable for detecting several types of
analytes (e.g. hormones or herbicides), it involves separation and
incubation steps in order to achieve desirable sensitivity.
[0012] Other immunoassays using electrochemical detection have to
rely on methods conventional in heterogeneous immunoassays, such as
lengthy incubation time and multiple washing steps to separate free
antigen and detection reagent from bound ones. Although several
groups have reported methods for performing non-separation
amperometric immunoassays, to date there have been no reports
describing an amperometric immunoassay that is simple, rapid, and
does not require a separation step.
[0013] Accordingly, there is still a need in the art for assay
devices and methods that provide simple, quantitative and real time
diagnostic measurements. The present invention addresses this other
related needs in the art.
BRIEF DESCRIPTION OF THE INVENTION
[0014] The present invention overcomes many of the problems in the
art by providing a simple, rapid, and reliable means to-measure,
and preferably, quantitatively measure, analyte in a liquid sample
using a combination of electrochemical detection and binding
between the analyte and its capture reagent.
[0015] In one aspect, the present invention is directed to a device
for detecting an analyte in a liquid sample, which device
comprises: a) a solid support; b) an electrosensor immobilized on
said solid support, said electrosensor comprises a working
electrode and another electrode used as auxiliary and/or reference
electrode; c) a capture reagent immobilized on said working
electrode, said capture reagent is capable of binding to an
analyte; and d) conductive leads for connecting said electrodes to
a readout device for electrochemical measurement.
[0016] The invention electrosensor, e.g., electroimmunosensor,
utilizes a sensor assembly (i.e., sensor strip). The sensor strip
can comprise a base sensor, e.g., a sensor fabricated by
screen-printing of conductive materials onto a suitable support. A
capture reagent that is capable of binding to the analyte is
immobilized on the working electrode surface. Preferably, the
capture reagent is capable of specific binding to the analyte,
which can be either an antibody specific to an epitope of the
analyte of interest, or the analyte of interest itself.
[0017] In addition to the sensor assembly, the device can
optionally has a sample application area and/or a detection area.
The detection area covers the region-of the electrode surface upon
which the capture reagent, e.g., antibody, is immobilized. The
application area can include an application pad having a detection
reagent pre-immobilized thereon. The detection reagent may be a
detection reagent, e.g., an antibody, labeled with an enzyme that
is able to produce an electrochemical detectable signal when
reacting with substrates.
[0018] The sensor assembly can also include a wicking member, e.g.,
in the form of a strip, that connects the application area and the
detection area. The wicking member functions as a carrier or
wicking reagent to deliver the fluid sample containing the analyte
and the detection reagent through capillary action to the detection
area where they become immobilized on the electrode surface
through, e.g., antibody-antigen reaction. Examples of materials
useful as the wicking member include nylon, cellulose, paper, and
the like. A preferred wicking member is a nylon mesh that has open
mesh structure. A mesh structure is particularly useful because it
has a two-dimensional structure suitable for lateral delivery of
the liquid sample from the application area to the detection area
to be in direct contact with the capturing reagent.
[0019] The sensor assembly may additionally include a conjugate
releasing pad for absorption and controlled release of a conjugate
and/or a separation filter for separating plasma from whole blood,
resulting in the plasma wicking laterally.
[0020] An absorbent material used as waste reservoir can be
positioned at the end of the sensor assembly, and overlaps with the
wicking member to facilitate the migration of the sample through
the device surface. The absorbent pad will have sufficient porosity
and volume to retain a liquid sample on which the assay is to be
performed.
[0021] In another aspect, the present invention is directed to a
method for assaying an analyte in a liquid sample, which method
comprises: a) contacting a liquid sample containing or suspected of
containing an analyte with the above-described device under
suitable conditions whereby the analyte, if there is any, binds to
the capture reagent immobilized on the working electrode and the
binding between the analyte and the capture reagent causes a change
in the current signal that is capable of being detected by the
electrosensor of the device; and b) detecting the change in the
current signal generated in step a), whereby the presence or amount
of the analyte in the sample is assessed.
[0022] In a preferred embodiment, the present method is used to
perform an enzyme immunoassay using the invention
electroimmunosensor. According to such a preferred method, a sample
containing the analyte of interest is applied to the sample
application area. The sample is allowed to flow through the
membrane assembled in the sensor strip to react with the antibody
immobilized on the sensor surface and with the antibody enzyme
conjugate. Under appropriate conditions, the analyte is sandwiched
between the, antibody immobilized on the sensor surface and the
antibody conjugate. The amount of analyte in the fluid sample is
proportional to the amount of analyte immobilized on the sensor by
this process through antibody-antigen interaction and can be
detected through the antibody-enzyme conjugate that is bound to the
sensor surface through the analyte. The amount of analyte is then
determined from a standard curve for the analyte of interest.
[0023] Detection can be achieved according to the invention method
by coupling the capture reagent-analyte binding reaction, e.g.,
immunological reaction, if the capture reagent used is antibody, to
an electrode response using the enzyme conjugated to the analyte
specific reagent as indicator. The current generated from a sensor
assembly under controlled conditions is proportional to the analyte
concentration present in the fluid sample and can be measured using
a electrocurrent detection device, e.g., an amperometric monitor.
Detection of analytes in buffer and serum samples can be
accomplished using the invention electrosensors, e.g.,
electroimmunosensors, in either competitive or sandwich assay
format.
[0024] Invention methods yield a rapid result by applying a test
sample to a disposable sensor strip and initiating electrochemical
detection when the invention electro-sensor is connected to an
electrochemical instrument, such as a hand-held detector. Good
sensitivity and quantitative results can be achieved within minutes
by unskilled personnel at point-of-care settings. Furthermore, the
methods can generally be used for quantitative measurement of
virtually any analytes, especially and immunologically active
species, in a liquid sample, thus providing broad application in
medical diagnostics and prognostics, and in agricultural and
environmental assessments. In addition, the electrosensor can be
provided in a kit suitable for use at home, in a physician's
office, or in other point-of-care settings.
[0025] In still another aspect, the present invention is directed
to a method for preparing an electrochemical sensor for the
detection of an analyte inca liquid sample, which method comprises
immobilizing a capture reagent capable of binding to an analyte on
the surface of a hydrophobic, non-metal electrode by contacting
said electrode surface with a solution containing said capture
reagent and an organic immobilizing agent that wets said electrode
surface and facilitates immobilization of said capture regent on
said electrode surface.
[0026] In yet another aspect, the present invention is directed to
a kit for detecting an analyte in a liquid sample, which kit
comprises: a) a device comprising a solid support, an electrosensor
immobilized on said solid support, said electrosensor comprises a
working electrode and another electrode used as auxiliary and/or
reference electrode, a capture reagent immobilized on said working
electrode, said capture reagent is capable of binding to an
analyte, and conductive leads for connecting said electrodes to a
readout device for electrochemical measurement; and b) an effective
amount of a suitable electron transfer mediator and substrate, and
any other buffer solutions, conjugate solutions or, standards
necessary for performing the detection assay.
[0027] In yet another aspect, the present invention is directed to
a device for detecting an analyte in a liquid sample, which device
comprises a sample application area that is in fluid communication
with an electrosensor via a wicking member, wherein the wicking
member has an open mesh structure.
BRIEF DESCRIPTION OF THE FIGURES
[0028] FIG. 1A is a schematic top view of an invention
electroimmunosensor.
[0029] FIG. 1B is a schematic top view of a base sensor 2 used in
an invention electroimmunosensor.
[0030] FIG. 2 is an exploded view of an invention
electroimmunosensor.
[0031] FIG. 3 is a graph showing the relationship of the amount of
prostate specific antigen in buffer (-.box-solid.-) and serum
(-.tangle-solidup.-) samples and the current response generated in
the detection area of an invention electroimmunosensor.
[0032] FIG. 4 is a graph showing the relationship between the
amount of alpha amino-fetoprotein (AFP) in a fluid sample and the
current response in the detection area using sensor strip
format.
[0033] FIG. 5 shows the electrochemical response of cardiac
troponin I in a fluid sample measured with an invention
electroimmunosensor.
[0034] FIG. 6 depicts a monitor for measuring electrocurrent.
[0035] FIGS. 7(A & B) depicts a disposable base sensor.
[0036] FIGS. 8(A & B) depicts a disposable sensor assembly.
[0037] FIG. 9 depicts a detection scheme using HRP.
[0038] FIG. 10 illustrates a sandwich assay format.
[0039] FIG. 11 shows troponin assay result. 11A shows effect of mAb
loading on sensor surface; 11B shows effect of membrane blocking on
non-specific signal; and 11C shows the assay result.
[0040] FIG. 12 shows several formats of sensor arrays that can be
used for base sensor for multi-analyte detection.
DETAILED DESCRIPTION OF THE INVENTION
A. Definitions
[0041] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of ordinary skill in the art to which this invention belongs. All
patents, applications, published applications and other
publications and sequences from GenBank and other databases
referred to herein are incorporated by reference in their entirety.
If a definition set forth in this section is contrary to or
otherwise inconsistent with a definition set forth in applications,
published applications and other publications and sequences from
GenBank and other data bases that are herein incorporated by
reference, the definition set forth in this section prevails over
the definition that is incorporated herein by reference.
[0042] As used herein, "a" or "an" means "at least one" or "one or
more."
[0043] As used herein, "a capture reagent" refers to any agent that
is capable of binding to an analyte. Preferably, "a capture
reagent" refers to any agent that is capable of specifically
binding to an analyte, i.e., having a higher binding affinity
and/or specificity to the analyte than to any other moiety. Any
moiety, such as a cell, a cellular organelle, an inorganic
molecule, an organic molecule and a mixture or complex thereof can
be used as a capture reagent so long that it has the desired
binding affinity and/or specificity to the analyte. The capture
reagent can be peptides, proteins, e.g., antibodies or receptors,
oligonucleotides, nucleic acids, vitamins, oligosaccharides,
carbohydrates, lipids, small molecules, or a complex thereof.
[0044] As used herein, "macromolecule" refers to a molecule that,
without attaching to another molecule, is capable of generating an
antibody that specifically binds to the macromolecule.
[0045] As used herein, "small molecule" refers to a molecule that,
without forming homo-aggregates or without attaching to a
macromolecule or adjuvant, is incapable of generating an antibody
that specifically binds to the small molecule. Preferably, the
small molecule has a molecular weight that is about or less than
10,000 daltons. More preferably, the small molecule has a molecular
weight that is about or less than 5,000 dalton.
[0046] As used herein, "vitamin" refers to a trace organic
substance required in certain biological species. Most vitamins
function as components of certain coenzymes.
[0047] As used herein, "lipid" refers to water-insoluble, oily or
greasy organic substances that are extractable from cells and
tissues by nonpolar solvents, such as chloroform or ether.
[0048] As used herein, a "receptor" refers to a molecule that has
an affinity for a given ligand. Receptors may be
naturally-occurring or synthetic molecules. Receptors may also be
referred to in the art as anti-ligands. As used herein, the
receptor and anti-ligand are interchangeable. Receptors can be used
in their unaltered state or as aggregates with other species.
Receptors may be attached, covalently or noncovalently, or in
physical contact with, to a binding member, either directly or
indirectly via a specific binding substance or linker. Examples of
receptors, include, but are not limited to: antibodies, cell
membrane receptors surface receptors and internalizing receptors,
monoclonal antibodies and antisera reactive with specific antigenic
determinants such as on viruses, cells, or other materials, drugs,
polynucleotides, nucleic acids, peptides, cofactors, lectins,
sugars, polysaccharides, cells, cellular membranes, and
organelles.
[0049] Examples of receptors and applications using such receptors,
include but are not restricted to:
[0050] a) enzymes: specific transport proteins or enzymes essential
to survival of microorganisms, which could serve as targets for
antibiotic [ligand] selection;
[0051] b) antibodies: identification of a ligand-binding site on
the antibody molecule that combines with the epitope of an antigen
of interest may be investigated; determination of a sequence that
mimics an antigenic epitope may lead to the development of vaccines
of which the immunogen is based on one or more of such sequences or
lead to the development of related diagnostic agents or compounds
useful in therapeutic treatments such as for auto-immune
diseases
[0052] c) nucleic acids: identification of ligand, such as protein
or RNA, binding sites;
[0053] d) catalytic polypeptides: polymers, preferably
polypeptides, that are capable of promoting a chemical reaction
involving the conversion of one or more reactants to one or more
products; such polypeptides generally include a binding site
specific for at least one reactant or reaction intermediate and an
active functionality proximate to the binding site, in which the
functionality is capable of chemically modifying the bound reactant
[see, e.g. U.S. Pat. No. 5,215,899];
[0054] e) hormone receptors: determination of the ligands that bind
with high affinity to a receptor is useful in the development of
hormone replacement therapies; for example, identification of
ligands that bind to such receptors may lead to the development of
drugs to control blood pressure; and
[0055] f) opiate receptors: determination of ligands that bind to
the opiate receptors in the brain is useful in the development of
less-addictive replacements for morphine and related drugs.
[0056] As used herein, "antibody" includes antibody fragments, such
as Fab fragments, which are composed of a light chain and the
variable region of a heavy chain. Antibody encompasses polyclonal
and monoclonal antibody.
[0057] As used herein, "labeled analyte" refers to labeled analyte,
or any fragment, derivative or analogue thereof that substantially
retains its binding affinity and/or specificity to the capture
reagent so that the labeled analyte, or any fragment, derivative or
analogue thereof, competes with unlabeled analyte in the sample
fluid in binding to the immobilized capture reagent.
[0058] As used herein, "a label that is capable of generating
electrocurrent under suitable conditions" refers to one or more,
but not all component(s) that is required for generating current
signal so that no electrocurrent can be generated in the absence of
such label and current signal can only be generated, even in the
presence of such a label, when other necessary current-generating
component(s) is provided. For example, horseradish peroxidase,
hydrogen peroxide and at least one electron transfer mediator(s),
such as ferrocene, or a derivative thereof, benzoquinone, ascorbic
acid or 3,3',5,5' tetramethylbenzidine, are needed to generate
electrocurrent. Any one or two, but not all three, of the
horseradish peroxidase, hydrogen peroxide and electron transfer
mediator can be used as such a label(s).
[0059] As used herein, "in fluid communication" means that liquid
can move from one part of the present device, e.g., a sample
application area, to another part of the device, e.g., an
electrosensor. The two or more parts of the device can be in fluid
communication by being physically linked together or adjacent to
each other, or the fluid communication can be mediated through
another part of the device, e.g., a wicking member.
[0060] As used herein, "a detection reagent" refers to any agent
that is necessary for generating a detectable current signal, which
can be used to assess the presence and/or quantity of the analyte
to be detected. The nature of a detection reagent is often
determined by the assay format. For a competitive assay, a
detection reagent can be a labeled analyte itself, or a fragment,
analogue or derivative thereof that substantially retains its
ability to bind to the analyte. In a competitive assay format, the
capture reagent must be capable of specifically binding to an
analyte. For a sandwich assay, a detection reagent can be a labeled
reagent that is capable of binding to an analyte. In a sandwich
assay format, at least one or both of the capture reagent and the
detection reagent must be capable of specifically binding to an
analyte.
[0061] As used herein, "a wicking member" refers to any substance
or material or a mixture or complex thereof that enables fluid
communication between different parts of the device by facilitating
capillary flow of the fluid.
[0062] As used herein, "a wicking member having an open mesh
structure" refers to woven or non-woven (extruded) fabrics made
from filament fibers that enable uniform openings to be produced.
Polyester, nylon, aramid, polyethylene, and glass fibers are among
the many fibers available that are suitable for the
applications.
[0063] As used herein the term "assessing" is intended to include
quantitative and qualitative determination in the sense of
obtaining an absolute value for the amount or concentration of the
analyte, e.g., a protein or nucleic acid, present in the sample,
and also of obtaining an index, ratio, percentage, visual or other
value indicative of the level of analyte in the sample. Assessment
may be direct or indirect and the chemical species actually
detected need not of course be the analyte itself but may for
example be a derivative thereof or some further substance.
[0064] As used herein, "nutrient or storage protein" refers to a
protein that is used by the cell as the nutrient source or storage
form for such nutrient. Non-limiting examples of nutrient or
storage proteins include gliadin, ovalbumin, casein, and
ferritin.
[0065] As used herein, "contractile or motile protein" refers to a
protein that endows cells and organisms with the ability to
contract, to change shape, or to move about. Nonlimiting examples
of contractile or motile proteins include actin, myosin, tubulin
and dynein.
[0066] As used herein, "structural protein" refers to a protein
that serves as supporting filaments, cables, or sheets to give
biological structures strength or protection. Non-limiting examples
of structural proteins include keratin, fibroin, collagen, elastin
and proteoglycans.
[0067] As used herein, "defense protein" refers to a protein that
defends organisms against invasion by other species or protect them
from injury. Non-limiting examples of defense proteins include
antibodies, fibrinogen, thrombin, botulinus toxin, diphtheria
toxin, snake venoms and ricin.
[0068] As used herein, "regulatory protein" refers to a protein
that helps regulate cellular or physiological activity.
Non-limiting examples of regulatory proteins include insulin,
growth hormones, corticotropin and repressors.
[0069] As used herein, "sample" refers to anything which may
contain an analyte for which an analyte assay is desired. The
sample may be a biological sample, such as a biological fluid or a
biological tissue. Examples of biological fluids include urine,
blood, plasma, serum, saliva, semen, stool, sputum, cerebral spinal
fluid, tears, mucus, amniotic fluid or the like. Biological tissues
are aggregates of cells, usually of a particular kind together with
their intercellular substance that form one of the structural
materials of a human, animal, plant, bacterial, fungal or viral
structure, including connective, epithelium, muscle and nerve
tissues. Examples of biological tissues also include organs,
tumors, lymph nodes, arteries and individual cell(s). The sample
may also be a mixture of target protein containing molecules
prepared in vitro.
[0070] As used herein, "expressed in a tissue or organ specific
manner" refers to a gene expression pattern in which a gene is
expressed, either transiently or constitutively, only in certain
tissues or organs, but not in other tissues or organs.
[0071] As used herein, "tissue" refers to a collection of similar
cells and the intracellular substances surrounding them. There are
four basic tissues in the body: 1) epithelium; 2) connective
tissues, including blood, bone, and cartilage; 3) muscle tissue;
and 4) nerve tissue.
[0072] As used herein, "organ" refers to any part of the body
exercising a specific function, as of respiration, secretion or
digestion.
[0073] As used herein, "plant" refers to any of various
photosynthetic, eucaryotic multicellular organisms of the kingdom
Plantae, characteristically producing embryos,
containingchloroplasts, having cellulose cell walls and lacking
locomotion.
[0074] As used herein, "animal" refers to a multi-cellular organism
of the kingdom of Animalia, characterized by a capacity for
locomotion, nonphotosynthetic metabolism, pronounced response to
stimuli, restricted growth and fixed bodily structure. Non-limiting
examples of animals include birds such as chickens, vertebrates
such fish and mammals such as mice, rats, rabbits, cats, dogs,
pigs, cows, ox, sheep, goats, horses, monkeys and other non-human
primates.
[0075] As used herein, "bacteria" refers to small prokaryotic
organisms (linear dimensions of around 1 .mu.m) with
non-compartmentalized circular DNA and ribosomes of about 70 S.
Bacteria protein synthesis differs from that of eukaryotes. Many
antibacterial antibiotics interfere with bacteria proteins
synthesis but do not affect the infected host.
[0076] As used herein, "eubacteria" refers to a major subdivision
of the bacteria except the archaebacteria. Most Gram-positive
bacteria, cyanobacteria, mycoplasmas, enterobacteria, pseudomonas
and chloroplasts are eubacteria. The cytoplasmic membrane of
eubacteria contains ester-linked lipids; there is peptidoglycan in
the cell wall (if present); and no introns have been discovered in
eubacteria.
[0077] As used herein, "archaebacteria" refers to a major
subdivision of the bacteria except the eubacteria. There are three
main orders of archaebacteria: extreme halophiles, methanogens and
sulphur-dependent extreme thermophiles. Archaebacteria differs from
eubacteria in ribosomal structure, the possession (in some case) of
introns, and other features including membrane composition.
[0078] As used herein, "virus" refers to an obligate intracellular
parasite of living but non-cellular nature, consisting of DNA or
RNA and a protein coat. Viruses range in diameter from about 20 to
about 300 nm. Class I viruses (Baltimore classification) have a
double-stranded DNA as their genome; Class II viruses have a
single-stranded DNA as their genome; Class III viruses have a
double-stranded RNA as their genome; Class IV viruses have a
positive single-stranded RNA as their genome, the genome itself
acting as mRNA; Class V viruses have a negative single-stranded RNA
as their genome used as a template for mRNA synthesis; and Class VI
viruses have a positive single-stranded RNA genome but with a DNA
intermediate not only in replication but also in mRNA synthesis.
The majority of viruses are recognized by the diseases they cause
in plants, animals and prokaryotes. Viruses of prokaryotes are
known as bacterophages.
[0079] As used herein, "fungus" refers to a division of eucaryotic
organisms that grow in irregular masses, without roots, stems, or
leaves, and are devoid of chlorophyll or other pigments capable of
photosynthesis. Each organism (thallus) is unicellular to
filamentous, and possesses-branched somatic structures (hyphae)
surrounded by cell walls containing glucan or chitin or both, and
containing true nuclei.
[0080] As used herein, "disease or disorder" refers to a
pathological condition in an organism resulting from, e.g.,
infection or genetic defect, and characterized by identifiable
symptoms.
[0081] As used herein, "infection" refers to invasion of the body
of a multi-cellular organism with organisms that have the potential
to cause disease.
[0082] As used herein, "infectious organism" refers to an organism
that is capable to cause infection of a multi-cellular organism.
Most infectious organisms are microorganisms such as viruses,
bacteria and fungi.
[0083] As used herein, neoplasm (neoplasia) refers to abnormal new
growth, and thus means the same as tumor, which may be benign or
malignant. Unlike hyperplasia, neoplastic proliferation persists
even in the absence of the original stimulus.
[0084] As used herein, cancer refers to a general term for diseases
caused by any type of malignant tumor.
[0085] As used herein, "an immune system disease or disorder"
refers to a pathological condition caused by a defect in the immune
system. The immune system is a complex and highly developed,
system, yet its mission is simple: to seek and kill invaders. If a
person is born with a severely defective immune system, death from
infection by a virus, bacterium, fungus or parasite will occur. In
severe combined immunodeficiency, lack of an enzyme means that
toxic waste builds up inside immune system cells, killing them and
thus devastating the immune system. A lack of immune system cells
is also the basis for DiGeorge syndrome: improper development of
the thymus gland means that T cell production is diminished. Most
other immune disorders result from either an excessive immune
response or an `autoimmune attack`. For example, asthma, familial
Mediterranean fever and Crohn disease (inflammatory bowel disease)
all result from an over-reaction of the immune system, while
autoimmune polyglandular syndrome and some facets of diabetes are
due to the immune system attacking self cells and molecules. A key
part of the immune system's role is to differentiate between
invaders and the body's own cells--when it fails to make this
distinction, a reaction against `self` cells and molecules causes
autoimmune disease.
[0086] As used herein, "a metabolism disease or disorder" refers to
a pathological condition caused by errors in metabolic processes.
Metabolism is the means by which the body derives energy and
synthesizes the other molecules it needs from the fats,
carbohydrates and proteins we eat as food, by enzymatic reactions
helped by minerals and vitamins. There is a significant level of
tolerance of errors in the system: often, a mutation in one enzyme
does not mean that the individual will suffer from a disease. A
number of different enzymes may compete to modify the same
molecule, and there may be more than one way to achieve the same
end result for a variety of metabolic intermediates. Disease will
only occur if a critical enzyme is disabled, or if a control
mechanism for a metabolic pathway is affected.
[0087] As used herein, "a muscle and bone disease or disorder"
refers to a pathological condition caused by defects in genes
important for the formation and function of muscles, and connective
tissues. Connective tissue is used herein as a broad term that
includes bones, cartilage and tendons. For example, defects in
fibrillin--a connective tissue protein that is important in making
the tissue strong yet flexible--cause Marfan syndrome, while
diastrophic dysplasia is caused by a defect in a sulfate
transporter found in cartilage. Two diseases that originate through
a defect in the muscle cells themselves are Duchenne muscular
dystrophy (DMD) and myotonic dystrophy (DM). DM is another `dynamic
mutation` disease, similar to Huntington disease, that involves the
expansion of a nucleotide repeat, this time in a muscle protein
kinase gene. DMD involves a defect in the cytoskeletal protein,
dystrophin, which is important for maintaining cell structure.
[0088] As used herein, "a nervous system disease or disorder"
refers to a pathological condition caused by defects in the nervous
system including the central nervous system, i.e., brain, and the
peripheral nervous system. The brain and nervous system form an
intricate network of electrical signals that are responsible for
coordinating muscles, the senses, speech, memories, thought and
emotion. Several diseases that directly affect the nervous system
have a genetic component: some are due to a mutation in a single
gene, others are proving to have a more complex mode of
inheritance. As our understanding of the pathogenesis of
neurodegenerative disorders deepens, common themes begin to emerge:
Alzheimer brain plaques and the inclusion bodies found in Parkinson
disease contain at least one common component, while Huntington
disease, fragile X syndrome and spinocerebellar atrophy are all
`dynamic mutation` diseases in which there is an expansion of a DNA
repeat sequence. Apoptosis is emerging as one of the molecular
mechanisms invoked in several neurodegenerative diseases, as are
other, specific, intracellular signaling events. The biosynthesis
of myelin and the regulation of cholesterol traffic also figure in
Charcot-Marie-Tooth and Neimann-Pick disease, respectively.
[0089] As used herein, "a signal disease or disorder" refers to a
pathological condition caused by defects in the signal transudation
process. Signal transudation within and between cells mean that
they can communicate important information and act upon it.
Hormones released from their site of synthesis carry a message to
their target site, as in the case of leptin, which is released from
adipose tissue (fat cells) and transported via the blood to the
brain. Here, the leptin signals that enough has been eaten. Leptin
binds to a receptor on the surface of hypothalamus cells,
triggering subsequent intracellular signaling networks.
Intracellular signaling defects account for several diseases,
including cancers, ataxia telangiectasia and Cockayne syndrome.
Faulty DNA repair mechanisms are also invoked in pathogenesis,
since control of cell division, DNA synthesis and DNA repair all
are inextricably linked. The end-result of many cell signals is to
alter the expression of genes (transcription) by acting on
DNA-binding proteins. Some diseases are the result of a lack of or
a mutation in these proteins, which stop them from binding DNA in
the normal way. Since signaling networks impinge on so many aspects
of normal function, it is not surprising that so many diseases have
at least some basis in a signaling defect.
[0090] As used herein, "a transporter disease or disorder" refers
to a pathological condition caused by defects in a transporter,
channel or pump. Transporters, channels or pumps that reside in
cell membranes are key to maintaining the right balance of ions in
cells, and are vital for transmitting signals from nerves to
tissues. The consequences of defects in ion channels and
transporters are diverse, depending on where they are located and
what their cargo is. For example, in the heart, defects in
potassium channels do not allow proper transmission of electrical
impulses, resulting in the arrhythmia seen in long QT syndrome. In
the lungs, failure of a sodium and chloride transporter found in
epithelial cells leads to the congestion of cystic fibrosis, while
one of the most common inherited forms of deaffiess, Pendred
syndrome, looks to be associated with a defect in a sulphate
transporter.
[0091] As used herein: stringency of hybridization in determining
percentage mismatch is as follows: (1) high stringency:
0.1.times.SSPE, 0.1% SDS, 65.degree. C.; (2) medium stringency:
0.2.times.SSPE, 0.1% SDS, 50.degree. C.; and (3) low stringency:
1.0.times.SSPE, 0.1% SDS, 50.degree. C. Equivalent stringencies may
be achieved using alternative buffers, salts and temperatures.
[0092] For clarity of disclosure, and not by way of limitation, the
detailed description of the invention is divided into the
subsections that follow.
B. Devices and Kits
[0093] In one aspect, the present invention is directed to a device
for detecting an analyte in a liquid sample, which device
comprises: a) a solid support; b) an electrosensor immobilized on
said solid support, said electrosensor comprises a working
electrode and another electrode used as auxiliary and/or reference
electrode; c) a capture reagent immobilized on said working
electrode, said capture reagent is capable of binding to an
analyte; and d) conductive leads for connecting said electrodes to
a readout device for electrochemical measurement.
[0094] Any suitable solid support can be used in the present
device. For example, plastic, polyvinyl chloride (PVC),
polyvinylidene fluoride (PVDF), paper, nylon, fiberglass,
polyethylene, nitrocellulose, a wicking member, e.g., a wicking
member having an open mesh structure, or a combination thereof can
be used as solid support in the present device.
[0095] The electrosensor of the present device must comprise a
working electrode and another electrode used as auxiliary and/or
reference electrode. In a specific embodiment, the electrosensor
can comprise a working electrode, an auxiliary electrode and a
reference electrode. In a preferred embodiment, the working
electrode and/or auxiliary electrode can comprise a screen-printed
carbon conductor and the reference electrode can comprise a
screen-printed silver or silver/silver chloride conductor.
[0096] Any capture reagent having desired binding affinity and/or
specificity to the analyte can be used in the present device. For
example, the capture reagent can be a cell, a cellular organelle,
an inorganic molecule, an organic molecule and a mixture
thereof.
[0097] Exemplary cells include animal, e.g., mammalian and human,
plant, fungus e.g., yeast, and bacterium cells. Exemplary cellular
organelles include nucleus, mitochondria, chloroplasts, ribosomes,
ERs, Golgi apparatuses, lysosomes, proteasomes, secretory vesicles,
vacuoles or microsomes, cytoplasm and other plasms within the such
cellular organelles.
[0098] The capture reagent can be macromolecules such as peptides,
proteins, e.g., antibodies or receptors, oligonucleotides, nucleic
acids, e.g., nucleic acids capable of hybridizing with the target
analyte nucleic acids under desired stringency, e.g., low, medium
or high stringency, vitamins, oligosaccharides, carbohydrates,
lipids, or small molecules, or a complex thereof.
[0099] Any proteins or peptides that are capable of binding, or
specifically binding, to an analyte can be used as the capture
reagent in the present device. For example, enzymes, transport
proteins such as ion channels and pumps, nutrient or storage
proteins, contractile or motile proteins such as actins and
myosins, structural proteins, defense protein or regulatory
proteins such as antibodies, hormones and growth factors can be
used.
[0100] Any nucleic acids, including single-, double and
triple-stranded nucleic acids, that are capable of binding, or
specifically binding, to analyte can be used as the capture reagent
in the present device. Examples of such nucleic acids include DNA,
such as A-, B- or Z-form DNA, and RNA such as mRNA, tRNA and
rRNA.
[0101] Any vitamins that are capable of binding, or specifically
binding, to analyte can be used as the capture reagent in the
present device. For example, water-soluble vitamins such as
thiamine, riboflavin, nicotinic acid, pantothenic acid, pyridoxine,
biotin, folate, vitamin B.sub.12 and ascorbic acid can be used.
Similarly, fat-soluble vitamins such as vitamin A, vitamin D,
vitamin E, and vitamin K can be used.
[0102] Any lipids that are capable of binding, or specifically
binding, to analyte can be used as the capture reagent in the
present device. Examples of lipids include triacylglycerols such as
tristearin, tripalmitin and triolein, waxes, phosphoglycerides such
as phosphatidylethanolamine, phosphatidylcholine,
phosphatidylserine, phosphatidylinositol and cardiolipin,
sphingolipids such as sphingomyelin, cerebrosides and gangliosides,
sterols such as cholesterol and stigmasterol and sterol fatty acid
esters. The fatty acids can be saturated fatty acids such as lauric
acid, myristic acid, palmitic acid, stearic acid, arachidic acid
and lignoceric acid, or can be unsaturated fatty acids such as
palmitoleic acid, oleic acid, linoleic acid, linolenic acid and
arachidonic acid.
[0103] In a preferred embodiment, the capture reagent is an
antibody, avidin/strepavidin, protein A or protein G.
[0104] The capture reagent can be immobilized on the working
electrode by any methods know in the art. Preferably, the capture
reagent is immobilized on the working electrode via a buffered
organic solution containing a ketone or an aliphatic alcohol, e.g.,
isopropyl alcohol.
[0105] Any analyte, e.g., cells, cellular organelles, inorganic
molecules, organic molecules and mixtures thereof can be detected
by the present device. Preferably, the analyte to be detected is
alpha-fetoprotein, prostate-specific antigen, cardiac troponins,
c-reactive protein (CRP), or human chorionic gonadotropin, or a
marker for HBV, HAV, HCV or HIV infection.
[0106] The device can further comprise an analyte bound to the
capture reagent, said analyte contains a label that is capable of
generating current signal under suitable conditions. When used, the
analyte, if there is any in the sample fluid, will bind to the
capture reagent immobilized and the binding between the unlabeled
analyte in the sample and the capture reagent displaces the labeled
analyte from the capture reagent and decreases current signal that
is capable of being detected by the electrosensor of the device.
The label can be an enzyme. Any enzyme that catalyzes a reaction
that leads to the generation of current signal under suitable
conditions can be used. In a specific embodiment, the enzyme is
horseradish peroxidase and the enzymatic substrate is hydrogen
peroxide and the electron transfer mediator is ferrocene, or a
derivative thereof, benzoquinone, ascorbic acid or 3,3',5,5'
tetramethylbenzidine.
[0107] The device can further comprise a cover casing having a
liquid sample application aperture and a detection aperture.
[0108] The device can further comprise a sample application area
that is separate, but in fluid communication with the
electrosensor.
[0109] The device can be used in both the competitive and sandwich
assay format. To be used in the competitive assay format, the
sample application area can contain deposited labeled analyte, said
labeled analyte is capable of being dissolved or suspended into the
sample liquid and being carried to the capture reagent immobilized
on the electrosensor by the sample fluid, and said label is capable
of generating current under suitable conditions.
[0110] To be used in the sandwich assay format, the sample
application area can contain deposited labeled detection reagent,
said labeled detection reagent is capable of being dissolved or
suspended into the sample liquid, binding to the analyte, if there
is any, being carried to the capture reagent immobilized on the
electrosensor by the sample fluid to form a sandwich comprising the
immobilized capture reagent-analyte-labeled detection reagent, and
said label is capable of generating electrocurrent current signal
under suitable conditions. Preferably, the deposited labeled
detection reagent is capable of specifically binding to the
analyte, if there is any, in the sample fluid. Also preferably, the
deposited labeled detection reagent is an antibody. The label can
be an enzyme. Any enzyme that catalyzes a reaction that leads to
the generation of electrocurrent under suitable conditions can be
used. In a specific embodiment, the enzyme is horseradish
peroxidase and the enzymatic substrate is hydrogen peroxide and the
electron transfer mediator is ferrocene, or a derivative thereof,
benzoquinone, ascorbic acid or 3,3',5,5' tetramethylbenzidine.
[0111] The sample application area must be in fluid communication
with the electrosensor. Preferably, the sample application area is
in fluid communication with the electrosensor via a wicking member.
Any suitable material or a mixture thereof can be used in the
wicking member. Preferably, the wicking member comprises nylon,
cellulose or paper. Also preferably, the wicking member comprises a
nylon mesh having mesh opening in the range from about 0.45 .mu.m
to about 100 .mu.m. Further preferably, the wicking member provides
for substantially two dimensional transport of fluids from the
application area to the electrosensor.
[0112] The device can further comprise a filter in the application
area, said filter is capable of removing insoluble or insuspendable
material(s) from the sample fluid. For example, the device can
comprise a filter that is adapted for removing insoluble or
insuspendable material(s) from a sample blood for the separation of
plasma or serum from blood.
[0113] The device can further comprise an absorptive sink in fluid
communication with the electrosensor, said sink having sufficient
porosity and capacity to absorb excess liquid or allow excess
liquid to be washed out of the device. The absorptive sink can use
any suitable material and in any suitable geometric patterns. For
example, the absorptive sink can be a pad of absorbent
material.
[0114] In a specific embodiment, the device comprises an absorptive
sink, an electrosensor and an application area that are linearly
arranged in order.
[0115] The device can further comprise an enzyme substrate and an
electron transfer mediator that are required for generating
electrocurrent that can be detected by the electrosensor.
Preferably, the enzyme substrate and the electron transfer mediator
are localized on or in proximity to the electrosensor, said
substrate and mediator can be controllably released.
[0116] In a specific embodiment, the present invention is directed
to a device for detecting an analyte in a liquid sample, which
device comprises: a) a base sensor strip having a working
electrode, a reference electrode, and an auxiliary electrode coated
on a plastic substrate, whereon a capture reagent is immobilized on
the working-electrode, said sensor strip having conductive leads
for attaching the electrodes to a readout device for
electrochemical measurement; b) a cover casing having a liquid
sample application aperture and a detection aperture; c) an
application zone for receiving a fluid containing an analyte from
the application aperture, said application zone, in the dry unused
form, containing a labeled detection reagent capable of
specifically binding to said analyte, wherein the said labeled
reagent is released into mobile form when in contact with the
liquid sample; d) a detection zone in fluid communication with the
electrodes in the presence of a liquid sample received from the
detection aperture; e) a wicking member that carries the liquid
sample from the application zone to the detection zone by capillary
action, wherein said analyte is sandwiched between the detection
reagent and the capture reagent immobilized on the electrode
surface; and f) an absorbent sink placed in partial contact with
the wicking member at the end of the flow path to absorb any excess
fluid from the detection zone.
[0117] In another specific embodiment, the present invention is
directed to a device for detecting an analyte in a liquid sample,
which device comprises a sample application area that is in fluid
communication with an electrosensor via a wicking member, wherein
the wicking member has an open mesh structure. Preferably, the
wicking member comprises a nylon mesh having mesh opening in the
range from about 0.45 .mu.m to about 100 .mu.m. Also preferably,
the wicking member provides for substantially two dimensional
transport of fluids from the application area to the electrosensor.
The device can further comprise a capture reagent capable of
binding to an analyte that is immobilized on the electrosensor.
[0118] In still another specific embodiment, the present invention
is directed to a kit for detecting an analyte in a liquid sample,
which kit comprises: a) a device comprising: 1) a solid support; 2)
an electrosensor immobilized on said solid support, said
electrosensor comprises a working electrode and another electrode
used as auxiliary and/or reference electrode; 3) a capture reagent
immobilized on said working electrode, said capture. reagent is
capable of binding to an analyte; and 4) conductive leads for
connecting said electrodes to a readout device for electrochemical
measurement; and b) an effective amount of a suitable electron
transfer mediator and substrate, and any other buffer solutions,
conjugate solutions or, standards necessary for performing the
detection assay.
C. Methods for Detecting Analytes
[0119] In another aspect, the present invention is directed to a
method for assaying an analyte in a liquid sample, which method
comprises: a) contacting a liquid sample containing or suspected of
containing an analyte with a device comprising: 1) a solid support;
2) an electrosensor immobilized on said solid support, said
electrosensor comprises a working electrode and another electrode
used as auxiliary and/or reference electrode; 3) a capture reagent
immobilized on said working electrode, said capture reagent is
capable of binding to an analyte; and 4) conductive leads for
connecting said electrodes to a readout device for electrochemical
measurement, under suitable conditions whereby the analyte, if
there is any, binds to the capture reagent immobilized on the
working electrode and the binding between the analyte and the
capture reagent causes a change in the electrocurrent that is
capable of being detected by the electrosensor of the device; and
b) detecting the change in the electrocurrent generated in step a),
whereby the presence or amount of the analyte in the sample is
assessed.
[0120] Any suitable capture reagent, including the capture reagents
that are described in the above Section B can be used in the
present method. Preferably, the capture reagent is an antibody.
Also preferably, the capture reagent is capable of specifically
binding to the analyte.
[0121] The present method can be used in both the competitive and
sandwich assay formats. In a specific competitive assay format, the
device used in the present method can comprise an analyte bound to
the capture reagent, said analyte contains a label that is capable
of generating current signal under suitable conditions, and the
binding between the unlabeled analyte in the sample and the capture
reagent displaces the labeled analyte from the capture reagent and
decreases current signal that is capable of being detected by the
electrosensor of the device. In an alternative competitive assay
format, the device used in the present method can comprise a sample
application area containing deposited labeled analyte, said labeled
analyte is capable of being dissolved or suspended into the sample
liquid and being carried to the capture reagent immobilized on the
electrosensor by the sample fluid, said label is capable of
generating electrocurrent under suitable conditions, and the
presence of unlabeled analyte in the sample fluid decreases current
signal that is capable of being detected by the electrosensor of
the device. In still another alternative competitive assay format,
the labeled analyte can be added in the sample fluid or can be
added separately, said labeled analyte is capable of being
dissolved or suspended into the sample liquid and being carried to
the capture reagent immobilized on the electrosensor by the sample
fluid, said label is capable of generating current signal under
suitable conditions, and the presence of unlabeled analyte in the
sample fluid decreases current signal that is capable of being
detected by the electrosensor of the device.
[0122] In a specific sandwich assay format, the device used in the
present method can further comprise a sample application area
containing deposited labeled detection reagent, said labeled
detection reagent is capable of being dissolved or suspended into
the sample liquid, binding to the analyte, if there is any, being
carried to the capture reagent immobilized on the electrosensor by
the sample fluid to form a sandwich comprising the immobilized
capture reagent-analyte-labeled detection reagent, and said label
is capable of generating current signal under suitable conditions.
Preferably, the deposited labeled detection reagent is capable of
specifically binding to the analyte, e.g., an antibody, if there is
any, in the sample fluid. In an alternative sandwich assay format,
a labeled detection reagent can be added in the sample fluid or can
be added separately, said labeled detection reagent is capable of
being dissolved or suspended into the sample liquid, binding to the
analyte, if there is any, being carried to the capture reagent
immobilized on the electrosensor by the sample fluid to form a
sandwich comprising the immobilized capture reagent-analyte-labeled
detection reagent, and said label is capable of generating current
signal under suitable conditions. Preferably, the added labeled
detection reagent is capable of specifically binding to the
analyte, e.g., an antibody, if there is any, in the sample
fluid.
[0123] The sample application area of the device used in the
present method must be in fluid communication with the
electrosensor. Preferably, the sample application area is in fluid
communication with the electrosensor via a wicking member. Any
suitable material or a mixture thereof can be used in the wicking
member. Preferably, the wicking member comprises nylon, cellulose
or paper. Also preferably, the wicking member comprises a nylon
mesh having mesh opening in the range from about 0.45 .mu.m to
about 100 .mu.m. Further preferably, the wicking member provides
for substantially two dimensional transport of fluids from the
application area to the electrosensor.
[0124] The device used in the present method can further comprise a
filter in the application area, said filter is capable of removing
insoluble or insuspendable material(s) from the sample fluid. For
example, the device can comprise a filter that is adapted for
removing insoluble or insuspendable material(s) from a sample
blood.
[0125] The device used in the present method can further comprise
an absorptive sink in fluid communication with the electrosensor,
said sink having sufficient porosity and capacity to absorb excess
liquid or allow excess liquid to be washed out of the device. The
absorptive sink can use any suitable material and in any suitable
geometric patterns. For example, the absorptive sink can be a pad
of absorbent material.
[0126] In a specific embodiment, the device used in the present
method comprises an absorptive sink, an electrosensor and an
application area that are linearly arranged in order.
[0127] The device used in the present method can further comprise
an enzyme substrate and an electron transfer mediator that are
required for generating electrocurrent that can be detected by the
electrosensor. Preferably, the enzymatic substrate and the electron
transfer mediator are localized on or in proximity to the
electrosensor, said substrate and mediator can be controllably
released. Alternatively, the enzyme substrate and the electron
transfer mediator are added in the sample fluid or is added
separately for generating current signal that is capable of being
detected by the electrosensor of the device.
[0128] The present method can be used to qualitatively or
quantitatively detect any analyte. Preferably, the analyte to be
detected is a marker for a biological pathway, a stage of cell
cycle, a cell type, a tissue type, an organ type, a developmental
stage, a disease, disorder or infection type or stage, or drug or
other treatments. Exemplary tissues include connective, epithelium,
muscle or nerve tissues. Exemplary organs include an accessory
organ of the eye, annulospiral organ, auditory organ, Chievitz
organ, circumventricular organ, Corti organ, critical organ, enamel
organ, end organ, external female gential organ, external male
genital organ, floating organ, flower-spray organ of Ruffini,
genital organ, Golgi tendon organ, gustatory organ, organ of
hearing, internal female genital organ, internal male genital
organ, intromittent organ, Jacobson organ, neurohemal organ,
neurotendinous organ, olfactory organ, otolithic organ, ptotic
organ, organ of Rosenmuller, sense organ, organ of smell, spiral
organ, subcommissural organ, subfornical organ, supernumerary
organ, tactile organ, target organ, organ of taste, organ of touch,
urinary organ, vascular organ of lamina terminalis, vestibular
organ, vestibulocochlear organ, vestigial organ, organ of vision,
visual organ, vomeronasal organ, wandering organ, Weber organ and
organ of Zuckerkandl can be manipulated. Exemplary internal animal
organs include brain, lung, liver, spleen, bone marrow, thymus,
heart, lymph, blood, bone, cartilage, pancreas, kidney, gall
bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous
system, gland, internal blood vessels. Exemplary diseases or
disorders include neoplasm (neoplasia), cancers, immune system
diseases or disorders, metabolism diseases or disorders, muscle and
bone diseases or disorders, nervous system diseases or disorders,
signal diseases or disorders, transporter diseases or disorders.
Exemplary infections include the infections caused by viruses,
bacteria or fungi. Preferably, the analyte to be detected is
alpha-fetoprotein, prostate-specific antigen, cardiac troponins,
c-reactive protein (CRP), or human chorionic gonadotropin or a
marker for HBV, HAy, HCV or HIV infection.
[0129] Analyte from any fluid sample can be detected by the present
method. Exemplary liquid sample include buffer, blood, serum,
plasma, or urine, or a solution or suspension containing solid
biological material.
[0130] In a specific embodiment, the present invention is directed
to a method for assaying an analyte in a liquid sample, which
method comprises: a) contacting a sample with a solution containing
a labeled detection reagent that specifically binds to an analyte
in the sample to form an assay mixture; b) incubating the assay
mixture with a device comprising a solid support, an electrosensor
immobilized on said solid support, said electrosensor comprises a
working electrode and another electrode used as auxiliary and/or
reference electrode, a capture reagent immobilized on said working
electrode, said capture reagent is capable of binding to an
analyte; and conductive leads for connecting said electrodes to a
readout device for electrochemical measurement, for a time period
sufficient for the analyte to become sandwiched between the labeled
detection reagent and the capture reagent immobilized on the
surface of the sensor; c) rinsing the electrosensor with an
appropriate buffer solution; d) adding a detection solution
containing a substrate and an electron transfer mediator to the
sensor surface to initiate an electron transfer reaction; and e)
determining current response generated from the electron transfer
mediator catalyzed by the labeled detection reagent, whereby the
presence or amount of analyte in the liquid sample is assessed.
[0131] In another specific embodiment, the present invention is
directed to a method for assaying an analyte in a liquid sample,
which method comprises: a) applying a fluid sample containing the
analyte of interest to the application zone of the device
comprising 1) a base sensor strip having a working electrode, a
reference electrode, and an auxiliary electrode coated on a plastic
substrate, whereon a capture reagent is immobilized on the working
electrode, said sensor strip having conductive leads for attaching
the electrodes to a readout device for electrochemical measurement;
2) a cover casing having a liquid sample application aperture and a
detection aperture; 3) an application zone for receiving a fluid
containing an analyte from the application aperture, said
application zone, in the dry unused form, containing a labeled
detection reagent capable of specifically binding to said analyte,
wherein the said labeled reagent is released into mobile form when
in contact with the liquid sample; 4) a detection zone in fluid
communication with the electrodes in the presence of a liquid
sample received from the detection aperture; 5) a wicking member
that carries the liquid sample from the application zone to the
detection zone by capillary action, wherein said analyte is
sandwiched between the detection reagent and the capture reagent
immobilized on the electrode surface; and 6) an absorbent sink
placed in partial contact with the wicking member at the end of the
flow path to absorb any excess fluid from the detection zone; b)
allowing the liquid sample to transport from application zone to
the detection zone by capillary action, wherein the analyte is
sandwiched between the labeled reagent and the capture reagent
immobilized on the sensor surface; c) adding a detection solution
containing a substrate and an electron transfer mediator through
the detection aperture to the detection zone to initiate an
electron transfer reaction; and d) amperometrically determining
current response generated from the electron transfer mediator
catalyzed by the labeled detection reagent, whereby the presence or
amount of the analyte in the liquid sample is assessed.
D. Methods for Preparing Electrochemical Sensors Containing Capture
Reagents
[0132] In still another aspect, the present invention is directed
to a method for preparing an electrochemical sensor for the
detection of an analyte in a liquid sample, which method comprises
immobilizing a capture reagent capable of binding to an analyte on
the surface of a hydrophobic, non-metal electrode by contacting
said electrode surface with a solution containing said capture
reagent and an organic immobilizing agent that wets said electrode
surface and facilitates immobilization of said capture regent on
said electrode surface.
[0133] Any suitable organic immobilizing agent can be used in the
present method. Preferably, the organic immobilizing agent is a
buffered aliphatic alcohol solution, e.g., isopropyl alcohol.
[0134] In a specific embodiment, the electrode is fabricated by
screen printing carbon composition upon a plastic substrate.
[0135] In another specific embodiment, the electrode is a working
electrode and is coupled with at least one additional electrode
fabricated by screen printing a conductive composition upon a
plastic substrate. Preferably, the working electrode and the
additional electrode are fabricated by screen printing carbon
composition upon the same plastic substrate.
[0136] Any suitable capture reagent, including the capture reagents
described in the previous Sections B and C, can be used in the
present method. For example, the capture reagent can be an amino
acid, a peptide, a protein, a nucleoside, a nucleotide, an
oligonucleotide, a nucleic acid, a vitamin, a monosaccharide, an
oligosaccharide, a carbohydrate, a lipid or a complex thereof.
Preferably, the capture reagent is an antibody, avidin/strepavidin,
protein A or protein G. Also preferably, the capture reagent is
capable of specifically binding to an analyte.
[0137] The method can further comprise coating the electrode
surface containing the immobilized capture reagent with a
stabilizing solution that stabilizes the immobilized capture
reagent. Preferably, the stabilizing solution stabilizes the
capture reagent immobilized on the electrode in a dry form. Any
suitable stabilizing solution can be used in the present method.
Preferably, the stabilizing solution contains a sugar, a
polyhydroxy compound, or StabilCoat.RTM..
E. Preferred Embodiments
[0138] In accordance with the present invention, there are provided
electroimmunosensors and methods for rapid and quantitative
measurement of the amount of various analytes in a variety of
liquid matrices. The invention electroimmunosensor comprises a base
sensor and a wicking member, which form a sensor assembly (i.e.,
sensor strip). See, for example, FIGS. 1 and 2.
[0139] As shown in FIG. 1B, base sensor 2 comprises support 4 with
working electrode 6, a reference electrode 8, and an auxiliary
electrode 10 printed thereon. Base sensors may be prepared in a
variety of ways, for example by screen-printing of a conducting
ink, such as carbon ink, on a large sheet of suitable support, such
as a sheet of Mylar.RTM. plastic, polyvinyl chloride (PVC), and the
like. The support sheet can then be cut to produce individual
sensors. The working electrode and the auxiliary electrode can both
be printed using carbon ink. The reference electrode is preferably
made using silver ink. To achieve better conductivity for the
working and the counter electrode, a layer of silver ink can
optionally be printed underneath the carbon ink. To provide an
insulating layer, a layer of dielectric film can also be printed
over of the conductive ink in the area of the printing that
corresponds to sample application area. Sensors prepared in this
manner can be used without further treatment for immobilization of
a capture antibody thereon.
[0140] A capture reagent specific to the analyte, for example a
primary antibody for a sandwich format immunoassay, is immobilized
onto the working electrode surface by any suitable means, for
example by spraying or spot casting. The capture reagent can be an
antibody specific to an epitope of the analyte of interest, or can
be the analyte of interest itself. The immobilized antibody is
preferably a monoclonal antibody with specificity of 10.sup.9
liters/mole, or greater, for the analyte of interest.
[0141] FIG. 1A shows a covering that fits over and protects the
base sensor 2. Cover 12 has two openings 14 and 16. When cover 12
is assembled with base sensor 2, openings 14 and 16 correspond to
and expose underlying application area 14' and detection area 16',
respectively, on the surface of the working electrode, which has
the. capture reagent immobilized thereon.
[0142] The three electrode leads shown in FIG. 1B form contacts for
connection of the electroimmunosensor strip to a potentiostat for
amperometric measurement of an electrochemically detectable species
on the working electrode surface. In the presence of a liquid
sample in the detection area 16', the electroimmunosensor functions
as an electrochemical cell. Thus when a constant potential is
applied to the working electrode with respect to the reference
electrode, a current passes between the working electrode and the
auxiliary electrode. The current flowing through the circuit is
directly proportional to the amount of the detection reagent
immobilized on the working electrode surface through the capture
antibody, as is described in details below. Commercially available
devices that can be used as potentiostat in accordance with the
invention include BAS Electrochemical analyzer (West Lafayette,
Ind.), Cypress System Electrochemical Analyzer (Lawrence, Kans.),
AndCare Electrochemical Monitor (Durham, N.C.) and the like.
[0143] Alternatively, an electroimmunosensor with two electrodes (a
working electrode and a reference electrode) can be used to replace
the 3-electrode system in accordance with the invention methods
when the solution resistance is negligible or the generated current
is relatively small (for example,. less than about 1 to about 5
.mu.A). In this case, one electrode, for example printed in carbon
ink, is used as the working electrode and the other electrode (for
example printed in silver or silver chloride ink) functions as both
reference electrode and auxiliary electrode. The working electrode
is immobilized with a capture reagent. The 2-electrode sensor can
be connected to an electrochemical device by conductive leads for
electrochemical measurement.
[0144] The sensor assembly according to the invention (as
illustrated, for example in FIG. 2) is designed for lateral flow of
sample and reagents, moving from left to right as shown in the
Figure. The sensor assembly comprises a wicking member to provide
for fluid flow from the application area 14 to the detection area
16. One important aspect of the present invention is the selection
of a wicking member. The primary function of an invention wick for
an electroimmunosensor is to act as a carrier for both the analyte
in the sample and the conjugate so that both are allowed to flow
through the wicking member and be captured on the electrode surface
through an antibody immobilized on the electrode surface.
Therefore, preferred attributes for a wicking member used in the
invention electroimmunosensor include low protein binding, good
flow characteristics, and water wetability. It is presently
preferred that the wicking member have a consistent flow rate and,
most importantly, that the flow is in a substantially lateral
direction. A lateral flow profile is more advantageous than a flow
profile containing both lateral and vertical directions. When a
sample fluid flows through a membrane laterally in a thin layer,
the analyte and conjugate are carried in closest proximity to the
antibody immobilized on a sensor surface, resulting in an enhanced
probability for formation of the antibody sandwich.
[0145] Membranes commonly used for other types of diagnostic tests,
such as nitrocellulose membranes, usually have a microporous
structure that generates flow in the vertical direction as well as
lateral flow. However, the three dimensional microporous structure
of membranes commonly used for other types of
membrane-based-immunoassay are disadvantageous for a lateral-flow
electroimmunosensor where the antibody is immobilized on the
surface of the sensor. Because the capture antibody is immobilized
on the surface of the sensor, the mass transport of the analyte and
the conjugate in the vertical direction generally limits the speed
of the assay. Any diffusion of the analyte and conjugate in the
vertical direction (as in microporous structures) does not
contribute to the formation of a sandwich, but may contribute to a
background signal caused by binding of the conjugate to the
membrane.
[0146] Wicking members having an open mesh structure ensure a
lateral flow profile while reducing the amount of non-specific
signal and are, hence, considered particularly advantageous for use
in the invention membrane-based electroimmunoassay. Another
consideration for lateral-flow immunoassay tests is the rate of
flow of fluids through the wicking material. Generally, a slow flow
rate enhances the assay signal but increases the assay time as
well. The wicking material having the most desirable set of
attributes for use in the present invention is nylon mesh having a
mesh opening sized in the range from about 10 .mu.m to about 100
.mu.m. A particularly suitable membrane material is nylon mesh made
of Nylon PA 6,6, for example, as manufactured by Millipore
(Bedford, Mass.) with open area up to 50% depending on mesh
opening. These materials are inherently hydrophilic, thus ensuring
instantaneous water wetting by capillary action without the use of
surfactants or other additives.
[0147] The sensor assembly also includes an absorbent material
(e.g., a pad) placed in partial contact with the wicking material
at the end of the flow path (e.g., the outside edge of the
detection area) to absorb and retain any excess fluid at the
leading end of the lateral flow path. Thus, the absorbent pad can
serve as a waste fluid reservoir. The absorbent material used in
the absorbent pad can be any water absorbent, porous medium that is
commercially available, such as Whatman absorbent paper, Grade
WF1.5 and F427-07, which are currently preferred for use as the
absorbent material in the absorbent pad.
[0148] The invention electroimmunosensor can further include a
conjugate pad having a detection reagent, such as a secondary
antibody labeled with an enzyme pre-immobilized thereon. The
conjugate pad is placed in contact with the wicking material. When
a sample is applied to the application area, the conjugate is
re-hydrated and carried through the wicking material. Thus, the
conjugate pad is particularly useful for conducting the
electroimmunoassay in sandwich assay format. Examples of materials
suitable for use as the conjugate releasing pad include
borosilicate glass fibers with a maximum of 5% PVA.
[0149] The sensor assembly may also optionally include a separation
filter through which fluids pass vertically (i.e., by wicking
action). The separation filter can be used, for example, to
separate plasma from whole blood.
[0150] The sensor assembly 5 (shown in exploded view in FIG. 2) is
also designed for lateral flow of reagents (from left to right as
shown in FIG. 2). In assembly 5 absorbent pad 18, wicking mesh 22
and conjugate pad 20 overlay the application area 14' and detection
area 16', with absorbent pad 18 being in contact with the extreme
leading edge of wicking strip 22 (shown to the left of the
detection area 16') and with conjugate pad 20 being in contact with
the opposite extreme edge of wicking strip 22. The detection
reagent, for example, an antibody labeled with (e.g., conjugated
with) an enzyme that is able to produce an electrochemical
detectable signal when reacting with a substrate and electron
transfer mediator, is immobilized on conjugate pad 20. Conjugate
releasing pad 20 is for absorption and controlled release of a
conjugate.
[0151] Wicking strip 22 connects the underlying application area
14' and the detection area 16' on the electrode surface and
functions as a carrier to deliver the fluid sample containing the
analyte and the detection reagent through capillary action to the
detection area 16' where the analyte will become immobilized on the
detection area of the electrode surface.
[0152] More specifically, when a sample containing an analyte is
added to the sensor assembly 5 through opening 14, the sample first
flows through the conjugate releasing pad 20 where contact with the
sample causes release of the antibody-enzyme conjugate deposited or
pre-immobilized on the conjugate releasing pad 20. The sample and
the released antibody-conjugate reagent are then further carried
through capillary action via wicking strip 22 past the detection
area 16' on the sensor surface where the analyte is captured in a
sandwich between the primary capture antibody immobilized thereon
and the antibody conjugate containing the secondary antibody.
Electrochemical Enzyme Immunoassays (EEIA)
[0153] Combining the sensitivity of electrochemical detection with
the selectivity and specificity of an immunoassay results in
extremely sensitive assays having a lower detection limit and wider
dynamic range than most other assay methods. Electrochemical
detection is particularly advantageous for use in immunosensors in
which the antigen-antibody reaction takes place on the surface of
an electrode. Electrochemical detection is usually accomplished by
amperometric detection of an electrode active species (electron
transfer mediator) catalyzed by an enzyme in the presence of its
substrate. See references: Ngo, T. T. Ed. Electrochemical Sensors
in Immunological Analysis, Plenum Press. New York, 1987; and
Monroe, D. "Amperometric Immunoassays" in Critical Reviews in
Clinical Laboratory Sciences. 28 (1): 1-18, 1990. Most commonly
used enzyme labels for EEIA include alkaline phosphatase (AP) and
horseradish peroxidase (HRP). In general, a desirable enzyme should
be able to efficiently catalyze an electron transfer reaction of a
suitable mediator in the presence of a substrate for the
enzyme.
[0154] In one embodiment according to the present invention, a
sandwich immunoassay format is used in which the enzyme horseradish
peroxidase (HRP) is conjugated to the second antibody used to form
the sandwich. Binding of an analyte specific to the immobilized
antibody determines the quantity of enzyme-conjugated antibody at
the electrode surface (and hence the amount of current generated by
the electrochemical reaction involved in the assay), thus
permitting the quantitation of the analyte of interest.
Alternatively, a competitive immunoassay format can be used in
which the enzyme horseradish peroxidase (HRP) is conjugated to the
analyte. In this case the analyte and the analyte HRP conjugate
compete for a limited number of binding sites on an antibody
immobilized electrode surface. Due to the competitive nature of the
assay, the amount of surface bound analyte-enzyme conjugate (and
hence the amount of current generated by the electrochemical
reaction involved in the assay) is inversely proportional to the
concentration of the analyte in the sample.
[0155] The activity of the enzyme is determined electrochemically
by the reduction of an electron transfer mediator. Examples of
mediators that may be used in the assays of the invention include
ferrocene and its derivatives, benzoquinone, ascorbic acid or
3,3',5,5' tetramethylbenzidine (TMB). TMB has been reported to be
suitable for use in ELISA with spectrophotometric measurement and
has been used as an electrochemical mediator for immunoassays where
HRP is used as the enzyme (G. Volpe et al., Analyst 123:1303-1307,
1998). TMB is found to be a good substrate for amperometric
determination of low levels of IRP and is preferred in the
invention.
[0156] Thus, in the practice of invention methods the surface bound
HRP conjugate is detected by adding preferred mediator TMB and
hydrogen peroxide. The concentrations of both TMB and hydrogen
peroxide are kept in excess in practice to ensure effective
enzymatic reaction. In the presence of hydrogen peroxide as a
co-substrate for HRP, TMB is oxidized and can then be reduced at a
relatively low potential.
[0157] By using a low potential, for example, within the range from
about 0 mV to about -200 mV (vs. Ag/AgCl), many biological
processes that commonly interfere with electrochemical assays do
not generate an interfering signal. The concentrations of the
electron transfer mediator and substrate used are usually kept in
excess of those required for the catalytic response of the enzyme.
Under this condition, a steady-state current from the recycling of
TMB on the electrode surface is generated for a given amount of HRP
conjugate. Because the solid phase on which the sandwich is formed
is also used for current measurement, for example by attachment of
electrical leads thereto, a steady-state current can be reached
within a few seconds after the addition of the substrate solution.
The current generated is proportional to the amount of HRP
conjugate bound to the electrode surface through the analyte. The
substrate solution can be added to the detection area (FIG. 1)
manually at the time of detection. Alternatively, the substrate
solution may be sealed in a reservoir, such as an alumna pouch and
prepackaged on the sensor or in fluid communication with the
detection area. In the latter case, the solution can be released at
the time of the assay by pressing the pouch or by controlled
rupture of the pack by mechanical means.
[0158] To perform an enzyme immunoassay using the disposable
invention electroimmunosensor, a sample containing the analyte of
interest is applied to the sample application area. A detection
reagent such as an enzyme antibody conjugate is released when in
contact with the sample. The sample and the detection reagent are
then allowed to flow through the wicking strip assembled in the
sensor strip to form a complex with the antibody immobilized on the
sensor. If a separation pad is present, for example for blood
separation, the pad is positioned on top of a conjugate pad and
facilitates separation of plasma from whole blood. Any excess of
the fluid sample wicking through the strip will be drawn to the
absorbent pad that serves as an absorbent sink. Under appropriate
conditions, the analyte is sandwiched between the antibody
immobilized on the sensor surface and the antibody conjugate. The
amount of analyte immobilized from the fluid sample is proportional
to the amount of analyte immobilized on the sensor through
antibody-antigen interaction and can be detected through the
antibody-enzyme conjugate that is bound to the sensor surface
through the analyte.
[0159] Alternatively, an immunoassay can be performed using a base
sensor where a capture antibody is immobilized on the working
electrode. In this embodiment of the invention assay methods, a
sample containing analyte of interest is mixed with an antibody
enzyme conjugate. The mixture is applied to the sensor and
incubated for a short time sufficient to allow formation of a
complex between analyte and antibody enzyme conjugate in the sample
and capture of the complex by the capture antibody, for example,
for about 5 to about 30 minutes.
[0160] After the incubation, the sensor is rinsed with buffer
solution to wash off unbound analyte and conjugate. The amount of
immobilized analyte is then detected by addition of detection
solution containing an enzyme substrate and an electron transfer
mediator. A steady-state current generated in the electrode from
the electrochemical reaction catalyzed by the enzyme is measured
using a conventional current read-out device. The amount of current
is proportional to the amount of analyte present in the sample
solution.
[0161] In accordance with another embodiment of the present
invention, test kits are provided for conducting a quantitative
electroimmunoassay, for example using an invention
electroimmunosensor. The invention kits comprise (a) a disposable
sensor strip having at least one test area, in which a capture
reagent is immobilized on the surface of the of the working
electrode. The kit may further include other components, such as a
hand-held monitor, standards for the analyte, buffer,solutions and
the like.
[0162] As used herein, the term "analyte" is defined broadly to
include any species or moieties. The present invention is
particularly applicable to virtually any analyte that generates
antibody-antigen reaction. Representative examples of types of
analytes include drugs, hormones, proteins, bacteria, viruses, and
cancer markers, and the like. Illustrative examples of analytes
that can be detected using the invention electroimmunosensors-and
methods include prostate specific antigen for prostate cancer
detection; alpha-fetoprotein (AFP) and human chorionic gonadotropin
(HCG) as markers for prenatal genetics screening, troponin I as
acute myocardial infarction marker, and the like. The analyte may
be determined in various liquid samples, including for example,
serum, blood, urine, saliva, and the like.
[0163] Although antibodies are used herein as an example of an
anti-analyte reagent since they are well characterized and
understood, the anti-analyte reagent used in the invention methods
and electroimmunosensor need not be limited to proteins and may be
another type of macromolecule, whether naturally occurring,
recombinant or synthetic, for example a synthetic receptor, a
carbohydrate/protein complex or nonprotein moiety, to which a
ligand or cross-reacting compound of interest will bind.
Nucleic Acid Detection
[0164] The device and the method can be adapted to a variety of
target amplification techniques for the detection of amplified
products. The commonly used amplification techniques include
polymerase chain reaction (PCR) for DNA target amplification, and
reverse transcriptase-PCR (RT-PCR), as well as isothermal nucleic
acid amplification systems including nucleic acid sequence-based
amplification (NASBA), the transcription-based amplification system
(TAS), transcription mediated amplification (TMA), and the ligase
chain reaction (LCR) system.
[0165] The detection of the amplified target can be achieved by
hybridization of the target with a capture probe and a detector
probe complementary to the target nucleic acid sequence, followed
by electrochemical detection of the detector probe. To perform the
assay, for example, a solution containing biotinylated detection
probe and a fluoresceinated oligonucleotide probes can be added to
the amplified reaction mixture. The capture probe and detection
probe will specifically bind to target DNA or RNA molecules. The
mixture solution can then be applied to a sensor containing a
pre-coated Avidin-HRP or streptavidin-HRP conjugate as a detector
reagent, and an anti-fluorescein antibody as a capture reagent.
When the mixture solution flows through the sensor via capillary
effect, the biotinylated portion of the probe will bind to the
streptavidin conjugate pre-coated on a conjugate pad, while the
fluoresceinated portion of the probe will be captured by the
antifluorescein antibody immobilized on the working electrode. The
HPR is detected electrochemically through a readout device. It
should be kept in mind that while the basic format is generic for
the detection of DNA/RNA target from an amplification technique,
variations in assay design exist for different applications.
[0166] A particular advantage of the present invention is that
quantitative assays can be performed by unskilled personnel
requiring no more steps than adding sample solution or detection
reagent. No lengthy incubation and sample separation are needed,
and the whole assay can be performed within minutes.
[0167] The invention will now be described in greater detail by
reference to the following non-limiting examples.
EXAMPLE 1
Preparation of Sensors by Printing
[0168] Base sensors as shown in FIG. 1 maybe purchased from a
commercial source or fabricated by screen printing of conductive
materials onto a suitable support, such as a plastic. The sensors
used in the experiments were printed using a polyester screen and
procedure recommended by the ink manufacture. The ink materials
include silver conductor and carbon conductor (Polymer Thick film
Compositions, 5000 and 7102, DuPont, Research Triangle Park, N.C.)
and the support was Mylar.RTM. plastic. The working electrode and
the auxiliary electrodes were both printed from carbon ink and the
reference electrode was printed from silver ink on Mylar.RTM.
plastic. To achieve better conductivity for the working and the
counter electrode, a layer of silver ink was printed underneath the
carbon ink. A layer of dielectric film can also be printed on top
of the portion of the conductive leads to provide an insulating
layer. Sensors prepared using such techniques can be used without
further treatment for immobilization of a capture antibody.
EXAMPLE 2
Antibody Immobilization on Screen-Printed Sensor
[0169] This example describes methods for immobilizing antibody on
a screen-printed sensor surface using aliphatic alcohol solutions.
It has long been know that certain organic solvents, including
alcohols and ketones, have stabilizing effect on proteins at low
concentrations, although these same organic solvents denature
proteins at high concentrations (Adachi and Schwartz, J. Biol.
Chem., 253(28):6423-6425 (1978)). The presence of alcohols or
ketones helps to wet the electrode surface and reduce static
charge, thus facilitating the attachment of the protein to the
surface.
[0170] Antibody was directly immobilized on carbon sensor surface
by applying a buffered antibody solution (such as PBS) containing
aliphatic alcohol. A preferred alcohol is iso-propanol. To
immobilize an antibody on a screen-printed sensor, 3 .mu.l of a
capture antibody solution in 10 mM phosphate buffer containing
iso-propanol was drop-coated on the working electrode area. The
alcohol solution evaporates into the air at room temperature,
leaving a layer of antibody bound to the electrode surface. The
amount of antibody added to the sensor needs to be optimized for
each capture antibody. Generally, however, the loading of
sufficient antibody to provide about a monolayer of antibody
coverage on the sensor surface gives the best sensor response.
Antibody immobilized on the sensor surface by such a method
generally retains its biological activity for at least a short
period of time.
[0171] To further stabilize the antibody immobilized on sensor for
long term usage, the antibody-coated surface was allowed to
incubate in a solution containing 25% StabilCoat.RTM. (SurModics,
Inc., Eden Prairie Minn.) and 0.01% Tween 20 at room temperature
for 10 minutes. After the incubation, the solution was aspirated
and the sensor was dried thoroughly before packaging in an airtight
container with a desiccant.
[0172] The percentage of alcohol in the immobilization solution
also influences the significance of the response. Experiments were
conducted to compare the influence of the amount of iso-propanol
used in an antibody immobilization solution. A monoclonal
anti-alpha feto protein antibody in PBS buffer containing various
percentage of isopropanol (in v/v %) was immobilized on the base
sensor using the method described in the above section. The
prepared sensors were incubated with a mixture solution containing
200 ng/ml alpha-fetoprotein (AFP) and an AFP-HRP conjugate at room
temperature for 15 minutes. After the incubation the sensors were
rinsed with PBS pH 7.4/0.5% Tween 20. The current response
generated from the immobilized AFP-HRP on each sensor was measured
using the method described in Example 3 herein. Table 1 below shows
the result of the signal dependence on the percentage of
iso-propanol used in the antibody immobilization solution. It has
been found that a 25% isopropanol in PBS solution is generally
suitable, and therefore is presently preferred for immobilization
of most capture antibodies.
1 TABLE 1 Relative Response of Signal % isopropanol % Signal 0 47.7
5 51.5 10 58.4 20 70.1 25 100 30 64.5
[0173] Methanol, ethanol, and ethyl acetate were also found to be
suitable solvents for immobilizing antibody on the electrode.
However results of immobilization tests indicated that the
stability of sensors immobilized with antibody in ethanol solution
decreases more dramatically over time than stability of antibodies
immobilized in iso-propanol solution.
[0174] Techniques other than drop-coating can also be used for
antibody immobilization. For example, the immobilization of
antibody can be made by spraying an antibody solution on to the
electrode surface, followed by evaporation of the solution. By
spraying, the uniformity of antibody loading on the sensor can be
controlled by selection of solvent and spray conditions.
Furthermore, the spray casting technique is more suitable to mass
production of the sensor for antibody immobilization.
EXAMPLE 3
Electrochemical Detection of HRP Conjugate
[0175] Binding of an analyte specific to immobilized antibody by a
second antibody conjugated to an enzyme results in formation of a
sandwich that permits quantitation of the analyte of interest
through the enzyme conjugate. Activity of the enzyme can be
determined electrochemically by reduction of an electroactive
species (an electron transfer mediator) in the presence of a
substrate for the enzyme. In the present example, the enzyme
horseradish peroxidase (HRP) was conjugated to a second antibody.
The electron transfer mediators that may be used for the invention
include dimethylaminomethyl ferrocene, ascorbic acid, benzoquinone,
and 3,3',5,5'-tetramethylbenzidine (TMB). A preferred mediator for
amperometric determination of HRP activity is TMB. TMB has been
reported to be suitable for use in ELISA with spectrophotometric
measurement and has been used as an electrochemical mediator for
immunoassays where HRP is used as the enzyme G. Volpe et al, supra.
In tests of the sandwich assay format, TMB was found to be a good
substrate for electrochemical detection of low levels of HRP. An
optimized substrate comprised 40 .mu.M TMB in 0.1M sodium acetate
(pH 6.0) solution containing 5-10% dimeathylsulfoxide, and 0.01%
hydrogen peroxide as a co-substrate for HRP enzymatic reaction.
Alternatively, ready-to-use liquid substrate solution containing
TMB, buffer, and hydrogen peroxide can be obtained from commercial
sources. Examples of such ready-to-use substrate solutions include
K-Blue Substrate.RTM. Ready-to-Use (TMB) (Neogen Corporation,
Lexington, Ky.) and 1-Step.TM. Turbo TMB (Pierce, Rockford,
Ill.).
[0176] The enzyme activity of HRP immobilized on a sensor was
measured using a Petite-Ampere analyzer (Bioanalytical Systems,
Inc., West Lafayette, Ind.). After the addition of the HRP
substrate solution to a sensor connected to the monitor, a
potential of -50 mV was applied. The current generated from HRP
immobilized on the sensor was measured 5 seconds after the addition
of the substrate solution.
EXAMPLE 4
Wicking Material (Membrane) Selection
[0177] The rate of capillary flow of buffer through several types
of wicking materials was compared to determine the wicking
properties of various materials. Porous membranes from various
manufacturers were compared with nylon net filters having different
size mesh openings (Millipore Corporation, Bedford, Mass.).
Membranes were cut into 4 mm by 4.5 cm strips. Each membrane strip
was fixed on a plastic support using a thin slice of transparent
tape across one end of the membrane. A buffer solution (phosphate
buffered saline (PBS)/0.5% casein, pH 7.4) was applied to each test
membrane strip on the taped end. The time required for the fluid to
flow through 4 cm of the test membrane was recorded. As shown in
Table 2 below, the flow rate through porous membranes was generally
slower than through membranes with mesh structure. Furthermore,
within the range of mesh sizes tested, the flow of buffer through
the membrane was directly proportional to the size of the openings
in the mesh. While the actual speed of the test procedure will be
determined by the flow rate of the membrane material employed,
these tests showed that any of the membranes tested could be used
to provide a rapid test. In addition, all the membranes tested
showed consistent flow characteristics.
2 TABLE 2 Time for buffer to flow Membrane Type Pore size 4 cm
along membrane Durapore, type SV 5 .mu.m 3'26" Durapore, type SV
0.63 .mu.m 3'45" Sartorius, cellulose Nitrate 8 .mu.m 2'32"
Sartorius, cellulose acetate 8 .mu.m 2'59" Whatman, cellulose
nitrate 3 .mu.m 3'47" Whatman, cellulose nitrate 5 .mu.m 3'42" MESH
NYLON NET FILTER OPENING Millipore, Nylon net 11 .mu.m 1'33"
Millipore, nylon net 30 .mu.m 1'05" Millipore, nylon net 80 .mu.m
.sup. 49" Millipore, nylon net 100 .mu.m .sup. 40"
[0178] The performance of the test wicking materials (membranes) in
electroimmunosensor response was compared using each of the
membranes in an invention electroimmunosensor.
Anti-alpha-fetoprotein (AFP) antibody was immobilized on a sensor
using the procedure as described in the Example 2 above. As second
antibody for the sandwich assay, an anti-AFP-HRP conjugate was
deposited and dried on a conjugate pad. The conjugate pads and
sensor strips utilizing the various test membranes were prepared
using procedures described in Example 7 hereinbelow. All the
wicking materials used in this example were unblocked.
[0179] To each sensor strip was added 100 .mu.l of 200 ng/ml AFP in
PBS/0.5% Casein, pH 7.4 solution. The sample was allowed to flow
through the test membrane assembled in the sensor strip to react
with the antibody immobilized on the sensor surface and with the
antibody-HRP conjugate. Electrochemical signal was measured using a
TMB/H.sub.2O.sub.2 substrate solution. The background response from
nonspecific signal was compared with the response from the analyte
by adding buffer solution without analyte to a sensor strip. Table
3 below shows the results of these tests:
3 TABLE 3 Signal Background Membrane Type Pore size .mu.A .mu.A
Durapore, type SV 0.63 .mu.m 4.01 2.51 Sartorius, cellulose Nitrate
8 .mu.m 2.00 1.12 Sartorius, cellulose acetate 8 .mu.m 1.83 0.99
Whatman, cellulose nitrate 3 .mu.m 4.36 2.44 Whatman, cellulose
nitrate 5 .mu.m 0.97 0.55 Millipore, nylon net 30 .mu.m 3.03
0.47
[0180] These results indicate that membranes with porous structure
generally generate much higher background signal than wicking
materials with mesh structure. On the other hand, even though the
flow of the test solutions in wicking materials with mesh structure
was much faster than in membranes with porous structure (see Table
2), a significant signal was generated, indicative of more
efficient antibody-antigen binding conditions. These data
demonstrate that wicking materials with mesh structure resulted in
the lowest background signal and highest signal-to-noise (S/N)
ratio compared with porous membranes, proving that wicking
materials with mesh structure are presently preferred for use in
the invention electroimmunosensors and methods.
[0181] Although wicking materials with mesh structure, such as
nylon net, are a preferred material for use as the membrane in the
invention electroimmunosensor, it is to be understood that other
materials having similar structure or having porous structure may
also be used for producing similar effect when they are optimized
to reduce the nonspecific signal.
EXAMPLE 5
Selection of Membrane Blocking Agents to Reduce Non-Specific
Signal
[0182] The electroimmunosensor response can be further improved by
blocking the wicking strip used in a sensor strip. The membrane
strip is blocked for the following purposes, among others: 1) to
reduce non-specific binding of the antibody-conjugate or analyte to
the membrane surface, and 2) to improve re-wetting and storage
properties of the finished device. Nonspecific attachment to the
wicking material can normally be reduced by blocking with a protein
(e.g. casein or bovine serum albumin), surfactant (e.g., Tween 20,
or Triton X-100), or a polymer (e.g., polyvinyl alcohol).
[0183] A preferred blocking agent in the invention is a polymer
having a hydrophobic center block and hydrophilic end blocks with
the structure PEG-PPG-PEG (where "PEG" is poly(ethylene glycol) and
"PPO" is poly(propylene glycol). Examples of such polymers are such
as Pluronic.TM. and Poloxamer.TM.. The center blocks can adsorb
onto a hydrophobic surface with the end blocks extending from the
surface and waving freely like seaweed. The coverage of the
hydrophobic center and the action of the hydrophilic end blocks
effectively block the membrane surface and create a surface that
does not absorb proteins.
[0184] To examine the effect of blocking reagent on nonspecific
signal, experiments were conducted under conditions similar to
those under which electroimmunosensor assays are performed to
determine the amount of HRP-antibody conjugate retained on a
blocked wicking material. The selected blocking reagents include
(a) a PEG-PPG-PEG polymer (average M.sub.n 8,400), (b) Triton
X-100, (c) PVA (M.sub.w 13,00-23000), and (d) (Tween 20), all from
Aldrich Chemical Company, Inc., Milwaukee, Wis. Dry wicking strips
(nylon mesh, 30 .mu.m mesh opening, Millipore) were soaked in
solutions containing 1% of a blocking reagent, and allowed to
equilibrate overnight at 4.degree. C. without shaking. The wicking
strips were then dried at room temperature and cut into sections
(0.5 cm by 2 cm) and assembled on a base sensor immobilized with a
capture antibody against troponin I, together with a absorbent pad
and a conjugate pad containing 30 ng/pad of a pre-dried
anti-troponin I HRP conjugate. 150 .mu.L of PBS pH 7.4/0.5% casein
buffer was applied to the application area of each
electroimmunosensor and allowed to flow through. After 5 minutes,
three drops of a substrate solution containing TMB/H2O2 were
applied to the detection area. The current signal from each sensor
strip was measured electrochemically using the method described in
Example 3.
[0185] The result indicates that comparing to wicking strip without
blocking, all the blocking reagents showed some effect in reducing
the nonspecific signal due to the attachment of the HRP conjugate
to the wicking strip. The effectiveness of blocking is in the
order: PEG-PPG-PEG>>PVA>Triton X-100>Tween 20. The
amount of blocking reagent used in the blocking solution is also
found influencing the amount of nonspecific signal. In general, the
nonspecific signal decreases with the increase of the amount of
blocking reagent used in blocking the wicking strip.
EXAMPLE 6
[0186] Antibody-HRP Conjugation
[0187] Many antibody-HRP conjugates are commercially available.
Alternatively, an antibody-HRP-conjugate can be made using Pierce's
EZ-Link.TM. Plus Activated Peroxidase kit (Pierce Chemical,
Rockford, Ill.) and a procedure described as follows:
[0188] 1. Dissolve approximately 1 mg IgG into 0.5-1.0 ml phosphate
buffered saline.
[0189] 2. Reconstitute 1 mg of lyophilized EZ-Link.TM. Plus
Activated Peroxidase with 100 .mu.l of water and add to the IgG
solution.
[0190] 3. Immediately add 10 .mu.l of sodium cyanoborohydride
solution, composed of 5 M NaCNBH.sub.3 in 1 M NaOH.
[0191] 4. Incubate the solution at room temperature for one
hour.
[0192] 5. Add 20 .mu.l of quench buffer composed of 3M
ethanolamine, pH 9.0 and react at room temperature for an
additional 15 minutes.
[0193] 6. Dialyze the conjugate in PBS solution using a
DispoDialyzer from Spectrum with 100,000 molecular weight cut off
(MWCO) to remove free HRP from the conjugate solution.
[0194] 7. Add Piece's SuperFreeze.TM. Peroxidase Conjugate
Stabilizer to the conjugate solution and store the solution in the
refrigerator for long term storage.
EXAMPLE 7
Conditions for Conjugate Releasing
[0195] A variety of materials can be used as the conjugate
releasing pad in the invention electroimmunosensor assays, for
example, borosilicate glass fiber with polyvinyl acrylic binder or
materials with polyester matrixes. The attributes to be sought in
selection of the material for fabrication of the conjugate
releasing pad include low protein binding, consistent flow
characteristics, uniform release of the conjugate and the like. A
preferred conjugate releasing material is Loprosorb.RTM. (KK0141
Gelman Sciences, Ann Arbor, Mich.).
[0196] In assays described herein, the conjugate releasing pad is
prepared by drying antibody conjugates onto the releasing pad. The
conjugate can be applied to the conjugate releasing pad either by
spraying a solution of the conjugate from a spray bottle or by
manual addition of the conjugate solution to the conjugate
releasing material with a micropipette. In the present example, an
antibody-HRP conjugate solution is diluted in 20% buffered sucrose
solution for application to the conjugate releasing pad. The
conjugate pad is dried before being placed in contact with the
lateral flow membrane in the sample application area. When a liquid
sample is applied to the application area, the conjugate is
rehydrated and carried through the membrane.
EXAMPLE 8
Disposable Sensor Strip for Prostate-Specific Antigen Detection
[0197] Base sensors of the type shown in FIG. 2 can be used alone
for the detection of analytes in a variety of liquids by applying a
sample containing the analyte of interest directly to the detection
area where an antibody is immobilized. In the present example, such
a base sensor was used for the detection of prostate specific
antigen (PSA) by immobilizing a monoclonal anti-PSA antibody on the
sensor strip using methods described in Example 2 above. PSA in a
PBS pH 7.4/0.5% casein solution and serum sample were incubated
concurrently with a second monoclonal anti-PSA antibody-HRP
conjugate, resulting in the capture of the conjugate on the surface
of the base sensor. After a 10-minute incubation at room
temperature, the sensor strip was rinsed with a PBS/0.05% Tween 20,
pH 7.4 solution, followed by addition of a substrate solution
containing hydrogen peroxide and TMB. The electrical leads from the
sensor were attached to a read-out device (Petite Ampere,
Bioanalytical Systems Inc., West Lafayette, Ind.), and the
resultant current was measured 5 seconds after addition of the
substrate solution. As is shown in FIG. 4, the current response was
directly proportional to the amount of PSA present in the test
sample. Examples of analytes that can be detected in this way
include, but are not limited to, human chorionic gonadotropin
(hCG), c-reactive protein and alpha-fetoprotein (AFP).
EXAMPLE 9
Sandwich Assay using Disposable Sensor Assembly
[0198] A sandwich-type electroimmunosensor assay may also be
performed for the detection of alpha-fetoprotein (AFP) using a
disposable membrane sensor in accordance with the present
invention. In this example, 3 .mu.l of 15 .mu.g/ml anti-AFP-HRP
conjugate purified according to the method described by Boorsman,
D. M. et. al. (J. Histochem. Cytochem. 23:200-207, 1976) was
deposited on a low protein binding conjugate pad from Gelman
Sciences (Loprosorb, KK0141). The conjugate pad was allowed to dry
at room temperature for 20 minutes and stored in a humidity
controlled chamber overnight before assembly into a sensor strip.
Then 30 ng/sensor of a monoclonal anti-AFP was immobilized on the
sensors and the sensor was assembled as described above.
[0199] To perform an enzyme immunoassay using the disposable
electroimmunosensor, 200 .mu.l of a sample solution containing AFP
in PBS/0.5% casein, pH 7.4 was applied to the sample application
area. The sample was allowed to flow through the wicking membrane
in the sensor strip to react with the antibody immobilized on the
sensor surface and with the antibody enzyme conjugate. After a few
minutes, a 100 .mu.l/sensor of substrate solution containing
TMB/H.sub.2O.sub.2 was added to the detection area of the sensor.
The current was measured 5 seconds after the addition of the
substrate solution using a read-out device (Petite Ampere,
Bioanalytical Systems, Lafayette, Ind.) connected to the sensor
strip. As is demonstrated in FIG. 5, the signal detected in the
detection area was continuously increased as the concentration of
the analyte in the fluid sample is increased.
[0200] In a similar way, the electroimmunosensor can be used to
detect a cardiac marker troponin I (TnI) for the detection of
cardiac injury. The electroimmunosensor for the assay prepared
using the described methods is assembled with a base sensor, an
absorbent pad, a conjugate pad, and a nylon mesh (30 .mu.m
Millipore). In each sensor is immobilized about 45 ng of a
monoclonal on the surface of the working electrode; the conjugate
pad contains 30 ng of second antibody against Troponin I that is
conjugated to HRP. The membrane used in this example are
pre-blocked with 2% PEG-PPG-PEG (Mn 8400). To performing the assay,
150 .mu.l of a sample liquid containing TnI is applied to the
sensor in the application area. After 5 minutes, K-Blue
Substrate.RTM. Ready-to-Use TMB (Neogen Corporation, Lexington,
Ky.) was added to the detection area. The current was measured 5
seconds after the addition of the substrate solution using a
read-out device (Petite Ampere, Bioanalytical Systems, Lafayette,
Ind.) connected to the sensor strip. The result, shown in FIG. 5,
demonstrated that the amount of troponin I present in the liquid
sample solution can be measured quantitatively over the clinical
relevant range using the invention electroimmunosensor.
EXAMPLE 10
Development of A New Immunosensor for Clinical Diagnosis: A
Disposable System with Lateral-Flow Design and Amperometric
Detection
Introduction
[0201] The development of a rapid, sensitive, and separation-free
method for the analyses of various analytes has been a
long-standing goal for biosensor technology. We have developed a
novel immunosensor system that combines the advantages of the
specificity of immunoassay, the fast reaction time of lateral-flow
chromatographic membrane assay, and the sensitivity of
electrochemical detection.
[0202] The system is based on the principle of immunoassay coupled
with amperometric detection using an enzyme as an indicator. The
sensor system comprises screen-printed electrodes immobilized with
antibodies. A nylon mesh is used as wicking agent for sample
delivery via capillary action, and an absorbent sink is placed at
the end of the flow path to absorb excess sample fluid. Lengthy
incubation and separation steps are eliminated by the lateral flow
sensor design with membrane materials and dry reagents incorporated
on the sensor. The sensor system has high sensitivity and
specificity, and is packaged into convenient and miniaturized
device that can be used at the point-of-care settings.
Design of the System
[0203] The design of the immunosensor system includes the
development for (1) a monitor for amperometric signal detection,
(2) an antibody immobilized base sensor, and (3) a sensor assembly
containing base sensor, all the reagents in dry form, and means for
sample handling as well as reagent delivery.
Monitor
[0204] Characteristics of the monitor shown in FIG. 6 include: 1)
Measurement of current at fixed potential; 2) LCD Screen; 3)
Hand-held, battery powered monitor; 4) Push button to start
measurement; 5) 5 seconds reading; and 6) Software/internet
capability in development.
Disposable Base Sensor
[0205] Characteristics of the disposable base sensor shown in FIG.
7 include: 1) Screen printing technique using carbon and Ag/AgCl
inks; 2) Mass production at low cost; 3) Antibody immobilized on
sensor surface; 4) Multiple sensor formats; and 5) Multianalyte
capability.
Disposable Sensor Assembly
[0206] Characteristics of the disposable sensor assembly shown in
FIG. 8 include: 1) Incorporation of base sensor, dry reagents, and
other components; 2) Lateral flow of liquid; 3)
Immuno-concentration effect; 4) Self-contained case; 5)
Compatibility for a variety of sample matrixes.
Detection Scheme
[0207] Horseradish peroxidase (HRP) can be used in the system for
the amplification of electrochemical signal. The activity of HRP is
detected by a:potentiostat using an electron pathway as shown
below. HRP catalyzes the oxidation of TMB in the presence of
hydrogen peroxide. TMB is then reduced at the electrode resulting
in a current when the applied potential is at -200 mV. The
magnitude of the current is directly proportional to the amount of
HRP on the electrode surface.
Immobilization of Antibody
[0208] Antibody is immobilized on the working electrode of a sensor
using our novel immobilization method. Antibody-based sensors were
prepared by drop coating antibody solution, and followed by surface
antibody stabilization. The finished sensor surface has high
antigen binding capacity and long-term stability.
Sandwich Assay Format
[0209] The immunosensor assay is based on a sandwich principal
where an analyte forms a "sandwich" between two antibodies (FIG.
10). One antibody is immobilized on the working electrode of the
sensor and the other is conjugated to an enzyme. Each antibody
binds to a different part of the analyte, enabling both to bind
simultaneously.
The Lateral Flow Electroimmunosensor
[0210] The lateral flow electroimmunosensor is a disposable strip
containing a base sensor immobilized with capture antibody, and
incorporated with membranes and other reagents in dry form to carry
out the analysis. One of the novel approaches in this design is to
introduce a nylon mesh as a wicking agent to achieve a
separation-free measurement. The sensor strip has a sample well and
a detection well, and is designed for lateral flow of reagents and
sample. The resulting sensor is a separation-free, disposable test
strip that can be used for quantitative measurement of analytes in
various sample matrixes.
Immunosensor for Troponin I Measurement
[0211] Cardiac Troponin I (cTnI) is the most specific marker for
myocardial injury. The upper limit of normal range is 0.2 ng/ml.
The Clinical significant range is 0.2-200 ng/ml. Experiments were
conducted to optimize the assay conditions including the antibody
loading on the sensor surface and the blocking of membrane to
reduce non-specific signal.
[0212] Cardiac Troponin I (cTnI) assay procedure include the
following steps: 1) Apply 50 .mu.l of sample containing Troponin I
to the sample well on the lateral flow immunosensor; 2) After 5
minutes, apply 50 .mu.l of H.sub.2O.sub.2/TMB to the detection well
on the sensor; and 3) Press the button on the monitor. Within 10
seconds, the monitor displays the measured current. Other assay
conditions are: 1) Immobilized mAb:70 ng/sensor; 2) 40 ng
HRP-Ab/Conjugate pad; 3) 30 .mu.m Nylon mesh (blocked with 10%
sucrose, 5% BSA, 1% Triton/PBS); and 4) 50 .mu.L buffer or serum
sample.
[0213] The assay results are shown in FIGS. 11A and B. Attributes
of the system used in the above-described assay include: 1)
User-friendly instrumentation; 2) Portable and low cost; 3)
Sensitive, specific, and quantitative tests; 4) Rapid detection
(assay time in minutes); 5) Simple assay procedure; and 6)
Multi-analyte capability.
Conclusion
[0214] We have developed a new disposable immunosensor system.
Feasibility of this system has been demonstrated for the
quantitative measurement of human troponin I in serum sample. The
system has the advantages of simplicity, high sensitivity, and low
cost. Separation and lengthy incubation steps commonly necessary
for immunoassays are eliminated due to the novel design of the
immunosensor. The system characteristics allow the development of a
new tool for clinical diagnosis of a wide range of analytes at the
point-of-care settings.
[0215] While the invention has been described in detail with
reference to certain preferred embodiments thereof, it will be
understood that modifications and variations are within the spirit
and scope of that which is described and claimed.
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