U.S. patent application number 11/284097 was filed with the patent office on 2006-06-22 for biosensor apparatus and methods of use.
This patent application is currently assigned to LifeScan, Inc.. Invention is credited to Ronald Chatelier, Alastair McIndoe Hodges, Dennis Rylatt.
Application Number | 20060134713 11/284097 |
Document ID | / |
Family ID | 37762369 |
Filed Date | 2006-06-22 |
United States Patent
Application |
20060134713 |
Kind Code |
A1 |
Rylatt; Dennis ; et
al. |
June 22, 2006 |
Biosensor apparatus and methods of use
Abstract
Disclosed herein are methods and devices for detecting the
presence of an analyte of interest. A biosensor device can include
a reaction chamber and an electrochemical detection chamber. The
reaction chamber can include at least one immobilized binding site
and a probe conjugate adapted to bind to at least one of the target
analyte and the immobilized binding site, while the detection
chamber can include electrodes for detecting an electrochemical
reaction. If present, the target analyte in the fluid sample
results in a change in the amount of probe conjugate bound in the
reaction chamber, which can be detected electrochemically in the
detection chamber.
Inventors: |
Rylatt; Dennis; (Wheelers
Hill, AU) ; Hodges; Alastair McIndoe; (Blackburn
South, AU) ; Chatelier; Ronald; (Bayswater,
AU) |
Correspondence
Address: |
LIFESCAN/NUTTER MCCLENNEN & FISH LLP
155 SEAPORT BOULEVARD
BOSTON
MA
02210-2604
US
|
Assignee: |
LifeScan, Inc.
Milpitas
CA
|
Family ID: |
37762369 |
Appl. No.: |
11/284097 |
Filed: |
November 21, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10830841 |
Apr 22, 2004 |
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11284097 |
Nov 21, 2005 |
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10105050 |
Mar 21, 2002 |
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11284097 |
Nov 21, 2005 |
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Current U.S.
Class: |
435/7.92 ;
435/287.2 |
Current CPC
Class: |
G01N 33/543 20130101;
G01N 33/54326 20130101; G01N 33/53 20130101; G01N 33/5438 20130101;
Y10T 29/49124 20150115 |
Class at
Publication: |
435/007.92 ;
435/287.2 |
International
Class: |
G01N 33/537 20060101
G01N033/537; C12M 1/34 20060101 C12M001/34; G01N 33/53 20060101
G01N033/53; G01N 33/543 20060101 G01N033/543 |
Claims
1. A biosensor for use in detecting a target analyte in a fluid
sample, the device comprising: a reaction chamber including an
immobilized binding site and a probe conjugate, wherein the probe
conjugate comprises a binding partner adapted to bind to the
immobilized binding site; a detection chamber including electrodes
for detecting an electrochemical reaction in the detection chamber;
and a fluid passageway between the reaction chamber and the
detection chamber, wherein, the presence or absence of a target
analyte in the fluid sample results in a change in the amount of
probe conjugate bound in the reaction chamber, the change
detectable with an electrochemical reaction in the detection
chamber.
2. The biosensor of claim 1, wherein the immobilized binding site
is an antigen and the binding partner is an antibody.
3. The biosensor of claim 1, wherein the immobilized binding site
is an antibody and the binding partner is an antigen.
4. The biosensor of claim 1, further comprising a filling chamber
adapted to be filled via capillary action.
5. The biosensor of claim 4, wherein the capillary dimension of the
reaction chamber is smaller in size than that of the filling
chamber.
6. The biosensor of claim 1, wherein the detection chamber includes
a vent.
7. The biosensor of claim 6, wherein the vent can be opened by
piercing an outer layer of the device.
8. The biosensor of claim 6, wherein the vent can be opened by
removing a portion of an outer layer of the device.
9. The biosensor of claim 6, wherein the vent can be opened by
tearing along a perforation.
10. The biosensor of claim 1, wherein the reaction chamber includes
an opening to the atmosphere.
11. The biosensor of claim 1, wherein immobilized binding site and
the probe conjugate are positioned on different surfaces within the
reaction chamber.
12. The biosensor of claim 1, wherein the immobilized binding site
is positioned on at least one magnetic bead.
13. The biosensor of claim 12, wherein the at least one magnetic
bead is dried on to an internal surface of the reaction
chamber.
14. The biosensor of claim 12, further comprising a magnet.
15. The biosensor of claim 14 wherein the magnet is disposed so as
to move the at least one magnetic bead to be in intimate contact
with the probe conjugate after the fluid sample has been introduced
into the reaction chamber.
16. The biosensor of claim 1, wherein the probe conjugate includes
an enzyme.
17. The biosensor of claim 16, further comprising a mediator and an
enzyme substrate.
18. An biosensor for use in detecting a target antigen in a fluid
sample, the device comprising: a reaction chamber including at
least one immobilized binding site including a antibody and a probe
conjugate including an antibody adapted to bind to bound target
antigen; a detection chamber including electrodes for detecting an
electrochemical reaction in the detection chamber; and a fluid
passageway between the reaction chamber and the detection chamber,
wherein, the presence of a target antigen in the fluid sample
results in a reduction in the amount of probe conjugate bound in
the reaction chamber, the reduction being detectable with an
electrochemical reaction in the detection chamber.
19. A method of detecting a target analyte in a fluid sample, the
method comprising the steps of: delivering a sample to a biosensor
that includes a reaction chamber and a detection chamber; allowing
a reaction to proceed in the reaction chamber between an
immobilized binding site and a probe conjugate; moving the sample
into a detection chamber and electrochemically detecting the level
of probe conjugate, wherein the presence of a target analyte in the
sample results in an increase or a decrease in the amount of probe
conjugate detected in the reaction chamber.
20. The method of claim 19, wherein the immobilized binding site
includes a target antigen and the probe conjugate includes an
antibody adapted to bind to the target antigen.
21. The method of claim 19, wherein the probe conjugate includes an
enzyme.
22. The method of claim 19, wherein the sample is moved from the
reaction chamber to the detection chamber via capillary action.
23. The method of claim 19, wherein the step of moving the sample
includes opening a vent.
24. The method of claim 19, further comprising the step of
quantifying the amount of target antigen in the sample based
electrical signals received from the detection chamber.
Description
RELATED APPLICATIONS
[0001] This application claims priority as a continuation-in-part
to U.S. application Ser. No. 10/105,050, entitled "Direct
Immunosensor Assay," filed Mar. 21, 2002, and Ser. No. 10/830,841,
entitled "Immunosensor," filed Apr. 22, 2004, both of which are
hereby incorporated by reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] Conventional biomedical sensors, including immunoassays
based systems, have been used to report the presence and/or
concentration of a wide variety of analytes. Immunoassays are
generally classified into two categories: competition assays and
sandwich assays. In a competition assay, the antigen in the test
sample is mixed with an antigen-probe complex (commonly referred to
as a reporter complex) and the mixture then competes for binding to
the antibody. The probe may be a radioisotope, a fluorophore, or a
chromophore. In a sandwich immunoassay, the antigen in the test
sample binds to the antibody and then a second antibody-probe
complex binds to the antigen. In these prior art assay methods, one
or more washing steps are usually required. The washing steps
introduce complexity into the assay procedure and can generate
biohazardous liquid waste.
[0003] Immunoassays usually provide a user with either a
qualitative result (e.g., a "yes/no answer") obtained, most often
by a simple visual detection (e.g., color change), or a
quantitative result such as a concentration of an antigen. Most of
the quantitative methods involve expensive pieces of equipment,
such as scintillation counters (for monitoring radioactivity),
spectrophotometers, spectrofluorimeters (see, e.g., U.S. Pat. No.
5,156,972), surface plasmon resonance instruments (see, e.g., U.S.
Pat. No. 5,965,456), and the like. It would therefore be
advantageous to develop an immunoassay that is both inexpensive and
simple enough to use to be suitable for home or field use. Such an
biosensor would preferably require no centrifugation, dilution,
pipetting, washing, or timing steps, and would generate minimal
waste.
SUMMARY OF THE INVENTION
[0004] Disclosed herein are biosensor devices and methods for
detecting and/or quantifying an analyte of interest. In one
embodiment, a disposable assay device is provided for use in
detecting a target analyte in a fluid sample, the device including
a reaction chamber with an immobilized binding target and a probe
conjugate positioned therein. When a sample is introduced into the
reaction chamber a binding reaction occurs between the immobilized
binding target, probe conjugate, and/or target analyte (if
present). In one exemplary embodiment, if the target analyte is
present, it causes a change in the amount of probe conjugate that
is bound to the immobilized binding site. This change can be
detected in a detection chamber.
[0005] The immobilized binding target, probe conjugate, and target
analyte can include the variety of known ligands. For example, one
skilled in the art will appreciate that the immobilized binding
target, probe conjugate, and target analyte can include an antigen
or antibody, a hormone or neurotransmitter and a receptor, a
substrate or allosteric effector and an enzyme, lectins and
surgars, DNA or RNA structures, such as aptamers and their binding
species (including other DNA or RNA species or binding protein),
proteins, biotin and adivin or streptavidin systems, enzymes and
their substrates and inhibitors, lipid binding systems, and
combinations thereof. To facilitate understanding of the devices
and methods described herein, immunological ligands will be
described henceforth and the device will be referred to as an
immunosensor or a biosensor.
[0006] In one aspect, the detection chamber mentioned above
includes electrodes and electrochemical reagents. For example, an
electrochemical reaction in the detection chamber can be used to
determine if the amount of probe conjugate bound in the reaction
chamber has been increased or reduced by the presence of the target
antigen. The electrochemical reaction can also be used to determine
the analyte (e.g., target antigen) concentration based on the
concentration of the probe conjugate in the detection chamber.
[0007] In one embodiment, the immobilized binding site includes an
antibody adapted to bind to a target antigen and the probe
conjugate includes an antibody adapted to bind to a bound target
antigen. If a target antigen is present in a fluid sample, the
immobilized binding site can bind to one site on the target
antigen, and the probe conjugate can bind to another site on the
target antigen. The presence of a target antigen in the fluid
sample thus results in an increase in the amount of probe bound in
the reaction chamber and a reduction in the amount of probe
conjugate in the detection chamber.
[0008] In another embodiment, the immobilized binding site includes
a target antigen and the probe conjugate includes an antibody
adapted to bind to the target antigen. When a sample is introduced
into the reaction chamber, a target antigen, if present, will bind
with the probe conjugate. As a result, the presence of a target
antigen in the fluid sample results in a reduction in the amount of
probe conjugate bound to the immobilized binding site in the
reaction chamber and an increase in the amount of unbound probe
conjugate that can travel to a detection chamber. The reduction in
bound probe conjugate can be detected and/or quantified by an
electrochemical reaction in the detection chamber. Conversely, the
immobilized binding site can include an antibody and the probe
conjugate can include the target antigen. The presence of target
antigen in the fluid sample will similarly cause a reduction in the
amount of bound probe conjugate in the reaction chamber.
[0009] In one aspect, the immobilized binding sites and the probe
conjugate are intermixed in the reaction chamber. Alternatively,
the immobilized binding sites and the probe conjugate are
positioned separately. For example, immobilized binding sites can
be located on magnetic beads that are dried on a surface of the
reaction chamber. When a liquid sample is introduced into the
reaction chamber, a magnetic field can be used to keep the magnetic
beads and immobilized binding sites from moving into the detection
chamber.
[0010] In another embodiment disclosed herein, a method of
detecting a target antigen in a fluid sample is provided. The
method can include the steps of delivering a sample to an biosensor
device that includes a reaction chamber and a detection chamber.
The sample is allowed to react with immobilized binding sites and a
probe conjugate positioned within the reaction chamber. The sample
is then moved to a detection chamber, and the method further
comprises the step of electrochemically detecting the probe
conjugate in the detection chamber. The detection step allows a
user to determine if the target antigen is present in the sample
based on the level of probe conjugate detected in the detection
chamber. The method can also include the step of quantifying the
amount of target antigen in the sample based on electrical signals
received from the detection chamber.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] The invention will be more fully understood from the
following detailed description taken in conjunction with the
accompanying drawings, in which:
[0012] FIG. 1 is a top view of one embodiment of an biosensor
disclosed herein;
[0013] FIG. 2 is a cross-sectional view of the biosensor of FIG. 1
along line A-A';
[0014] FIG. 3 is a top view of another embodiment of a biosensor
disclosed herein;
[0015] FIG. 4A is a cross-sectional view of the biosensor of FIG. 3
along the line A-A';
[0016] FIG. 4B is a cross-sectional view of the biosensor of FIG. 3
along the line B-B';
[0017] FIG. 4C is a cross-sectional view of the biosensor of FIG. 3
along the line C-C'; and
[0018] FIG. 4D is a cross-sectional view of the biosensor of FIG. 3
along the line D-D'.
DETAILED DESCRIPTION OF THE INVENTION
[0019] Described herein are biosensor devices and methods of use.
In one embodiment, the sensor includes a reaction chamber and a
detection chamber. Positioned within the reaction chamber are
immobilized binding sites that bind to the analyte of interest or
to species related to the analyte of interest. Also in this chamber
is a probe species that can be detected in the detection chamber,
and which can be conjugated to a species that can bind to the
immobilized binding site, or which can bind to a species that binds
to the immobilized binding site. This will be hereafter termed the
probe conjugate. The probe conjugate and the immobilized binding
site are such that the presence of the analyte of interest in the
sample modifies the interaction of the probe conjugate and the
immobilized binding site. For example, when present, the analyte
can block the probe conjugate from binding to the immobilized
binding site. Alternatively, the analyte can provide a site for the
probe conjugate to bind, thereby increasing the amount of bound
probe conjugate. In either embodiment, the presence of analyte
modifies the amount of the probe conjugate bound in the reaction
chamber.
[0020] The reaction chamber can be arranged so that after the probe
conjugate binding reactions have taken place to the desired extent,
the liquid from the reaction chamber is transferred to the
detection chamber, transferring with it the free probe conjugate
and leaving behind the bound probe conjugate. In the detection
chamber, the amount of free probe conjugate can be detected. For
example, electrodes in the detection chamber can be used to
electrochemically detect the level of probe conjugate in the
detection chamber. The electrochemical reaction can determine if
the target analyte is present/absent and/or determine the
concentration of the target analyte based on the amount of the
probe conjugate in the detection chamber.
[0021] A first embodiment of a biosensor 20, illustrated in FIGS. 1
and 2, includes a detection chamber 28 comprising an
electrochemical cell and a reaction chamber 22 containing
immobilized binding sites and a probe conjugate. The detection
chamber 28 and reaction chamber 22 can be prepared by forming an
aperture extending through a sheet of electrically resistive spacer
material 36. The aperture can be shaped such that it defines a
sidewall of both the reaction chamber 22 and detection chamber 28,
as well as a sample passageway 38 between chambers 22, 28. By
extending the aperture from a proximal end 24 of reaction chamber
22 through to an edge 37 of sensor 20, a sample ingress 25 is also
formed. In one embodiment, the thickness of sheet 36 defines the
height of the reaction chamber 22 and detection chamber 28, and the
chambers can have an equal height. According to this embodiment the
capillary force in the detection chamber must be greater than that
in the reaction chamber. This can be achieved by modifying the
surfaces of the reaction chamber and/or detection chamber or by
adding filling materials, such as those herein disclosed, to the
detection chamber.
[0022] In another embodiment, the height of reaction chamber 22 is
greater than that of detection chamber 28. A reaction chamber 22 of
greater height than detection chamber 28 can be prepared, for
example, by layering multiple inner sheets 32, 34, 36 and/or outer
sealing sheets 42, 46 together. For example, in FIG. 2 the middle
sheet 36 of sensor 20 has an aperture defining the sidewalls of
reaction chamber 22 and detection chamber 28 as described above.
Middle sheet 36 is then sandwiched between one or more additional
layers 32, 34, the additional layers 32 and 34 having an aperture
corresponding only to reaction chamber 22. With respect to
detection chamber 28, layers 32 and 34 define the end walls 60, 62
(i.e., top and bottom surfaces) of the chamber. In this embodiment,
the end walls 60 and 62 of the detection chamber comprise
electrodes 54 and 52, electrically connectable, via connection
means, to a measuring circuit. The electrodes are described in more
detail below.
[0023] In one aspect, the electrodes 52 and 54 can be placed in
electrical connection with a meter (not shown) through the
connection end 66. The connection end allows a meter (not shown) to
electrically communicate with the electrodes 52 and 54 in the
detection chamber 28 via electrically conductive tracks (not
shown). The meter in connection with the connection area 66 is
capable of applying a potential between the electrodes 52 and 54 in
the detection chamber 28 and detecting the electrical signals
generated during an electrochemical reaction.
[0024] In use, a user first introduces sample into the first
chamber, the reaction chamber 22, of the sensor through sample
ingress 25. The sample can be drawn into the reaction chamber under
the influence of capillary or wicking action. The reaction chamber
can include a vent 26 that is open to the atmosphere, thus allowing
air displaced by the sample to escape. Sample will be drawn into
the first chamber until it is filled up to the reaction chamber
vent 26, whereupon filling will stop. The volume of reaction
chamber 22 is chosen so as to be at least equal to and preferably
larger than the volume of the detection chamber 28.
[0025] The dashed circle in FIG. 1 denotes an aperture 30 piercing
layers 32, 34, and/or 36 but not layers 42 and 46. Since layers 42
and 46 are not pierced initially, the only opening to the
atmosphere of the detection chamber 28 is through sample passageway
38 that opens from reaction chamber 22. Thus, when reaction chamber
22 fills with sample, air is trapped in detection chamber 28, which
substantially prevents it from filling with sample. A small amount
of sample can enter the detection chamber 28 during the time
between when the sample first contacts the opening 38 to the
detection chamber 28 and when it contacts the far side of the
opening 38. However, once the sample has wet totally across the
opening 38 to the detection chamber 28, no more filling of
detection chamber 28 will take place.
[0026] The opening of a vent 56 to the atmosphere allows the air
trapped in the detection chamber 28 to escape, thereby permitting
detection chamber 28 to be filled with reacted sample from reaction
chamber 22. Vent 56 can be opened in a variety of ways, including,
for example, by puncturing an outer layer of the device, by
removing a portion of the outer layer of the device, and/or by
tearing away a portion of the device.
[0027] When the vent is opened, the reacted sample will be drawn
into the detection chamber 28 due to increased capillary force in
the detection chamber 28 compared to that present in the reaction
chamber 22. In one embodiment, the increased capillary force is
provided by suitably coating the surfaces of the detection chamber
28 or, more preferably, by choosing the capillary distance for
detection chamber 28 to be smaller than that of reaction chamber
22. In this embodiment, the capillary distance is defined to be the
smallest dimension of the chamber. One skilled in the art will
appreciate that the capillary forces in the reaction and/or
detection chambers can be created by varying a number of factors.
Capillary forces in thin chambers are discussed, for example, in
U.S. Pat. No. 6,823,750, entitled "Method of Preventing of
Preventing Short Sampling of a Capillary or Wicking Fill Device,"
which is hereby incorporated by reference in its entirety.
[0028] A second exemplary embodiment of a biosensor 120, including
three chambers, is illustrated in FIGS. 3 through 4D. The
immunosensor can include a filling chamber 107 in addition to a
reaction chamber 122 and a detection chamber 128. Sensor 120 can be
formed from multiple layers as described above, including for
example, a sealing layer 142, a lower layer 134, a spacer layer
136, and an upper layer 132. In one aspect, each layer comprises an
insulating material, while upper and lower layers 132, 134
additionally include an electrically conductive film as discussed
in more detail below. By removing portions of the layers at
different points in the sensor, a filing chamber 107, reaction
chamber 122, and a detection chamber 128 are formed. In addition,
exposing portions of the electrically conductive film on the upper
and lower layers 132, 134 provides electrodes 152, 154 for
performing electrochemical reactions and provides electrical
contact areas 101, 102, 103 for electrically connecting the sensor
to a meter.
[0029] Filling chamber 107 receives the sample from the patient or
user and provides a reservoir of sample for filling the other two
chambers. Reaction chamber 122 and detection chamber 128 are in
fluid communication with filling chamber 107. To assist with moving
fluid between chambers, detection chamber 128 can include vent 130
which is initially closed. After a sample is reacted in the
reaction chamber, vent 130 is opened so that air in detection
chamber 128 can exit through the vent allowing liquid from reaction
chamber 122 to enter the detection chamber. As discussed above with
respect to vent 56, vent 130 can be opened in a variety of ways,
including piercing the device, removing an outer layer, and/or
tearing a portion of the device (i.e., tearing along a
perforation).
[0030] The sensor can include electrical connection point 101 that
allows an electrical connection to be made to a lower electrode 152
and electrical connection points 102, 103 that allow an electrical
connection to an upper electrode 154. Dotted line 106 denotes a
break in the electrically conductive film defining upper electrode
154 on upper layer 132. The break may be affected by patterning the
conductive film when it is laid down or by creating the break
during manufacture. The break could be affected by scratching the
film, scraping part of the film away, chemically etching the film,
laser ablating the film or other methods as commonly known. Break
106 in the conductive film serves to, in part, define the active
electrode area of the strip by electrically isolating the
conductive coated in the detection chamber from that in the
reaction chamber. This is advantageous as it can prevent any
electric signal that might otherwise flow at the conductive films
in the reaction chamber from effecting the test results.
[0031] Sensor 120 can also include contact point 103 which allows a
user to electrically connect to the portion of conductive film in
contact with reaction chamber 122. As reaction chamber 122 fills
with sample, monitoring contact point 103 allows a signal to be
detected that indicates to the meter that the strip has been
successfully filled and a test sequence can commence. In order to
accomplish this with the embodiment of the invention shown in FIGS.
3 through 4D, an electrical connection could be made to the lower
conductive film at contact point 101 and to the upper conductive
film in the reaction chamber at contact point 103. A potential
would then be applied between the two connection points (101, 103)
and the current, voltage, and/or electrical resistance monitored to
ascertain when sample has entered the reaction chamber. This
potential may be a DC potential or it may be a potential that
varies with time such as an AC potential or a series of square wave
potential pulses with alternating polarity. By monitoring the
current that flows as a result of the potential application, or the
voltage required to pass a pre-determined current, an indication of
when the conductive films in the reaction chamber begin to wet can
be obtained.
[0032] Connection area 101, for electrically contacting a lower
layer 134 carrying the lower conductive film, can be formed by
extending lower layer 134 out past the end of a spacer layer 136
and the upper layer 132. Contact area 102 is formed by removing
sections of layers 134 and 136 to expose a section of upper layer
132. Contact area 103 is similarly formed by removing a section of
lower layer 134 and spacer layer 136 as shown in FIG. 4D
(cross-section D-D' In FIG. 3).
[0033] Filling chamber 107 can be formed by removing sections of
lower layer 134 and spacer layer 136, but leaving upper layer 132
and sealing layer 142 intact. Sealing layer 142 can be adhered to
the outside face of layer 134 and can serve, with the sides of the
cut-out sections in layers 134 and 136 and layer 132, to form a
capillary channel which is capable of drawing sample into it by
capillary action. This channel is illustrated in FIG. 4A
(cross-section A-A' in FIG. 3).
[0034] Reaction chamber 122 is formed by removing a section of the
spacer layer 136 but leaving layers 134 and 132 intact. This forms
a capillary space where the height of the capillary spacer is
smaller than the height of the filling chamber 122. This allows
capillary forces to draw liquid from the filling chamber 122 into
the reaction chamber 128 by capillary action. The small height of
the reaction chamber can also allow for relatively rapid mixing of
components in the reaction chamber. In one aspect, reaction chamber
122 opens at the lateral edge(s) of the strip to allow air to vent
while liquid fills the reaction chamber.
[0035] Detection chamber 128 is formed in a similar fashion to the
reaction chamber 122 by removing a section of the spacer layer 136
while leaving the layers 134 and 132 intact. Initially, the
detection chamber 128 opens to the reaction chamber 122 at one end
but has no other opening.
[0036] Vent hole 130 is incorporated into the detection chamber 128
by removing sections of or piercing upper layer 132 (or lower layer
134). A layer 146 shown in FIG. 4B (cross-section B-B' in FIG. 3)
can be laminated to the upper face of the strip to seal off the
opening. Alternatively, if a portion of lower layer 134 is removed,
sealing layer 142 can be pierced/removed to open vent hole 130.
[0037] When liquid sample fills reaction chamber 122, as part of
the filling process it will bridge the opening of detection chamber
128. Thus, when liquid fills reaction chamber 122 such that it runs
across the opening of detection chamber 128, air is trapped in
detection chamber 128 preventing further liquid from entering. This
allows liquid to be held in reaction chamber 122 while the binding
reactions are proceeding. After a pre-determined time when any
binding reactions that might be occurring in reaction chamber 122
have proceeded to the desired extent, layers 142 or 146 are pierced
by a piercing means (or removed/torn away) to allow air to escape
detection chamber 128, such that liquid transfers from reaction
chamber 122 to detection chamber 128. The cross-sectional dimension
of detection chamber 128 allow capillary action to fill the
detection chamber.
[0038] One skilled in the art will appreciate that the immunosensor
described herein can have a variety of alternative configurations
such as, for example, the shape of the sensor, the number of
chambers, the electrode configuration, and/or the placement of
electrical contact points. For example, other sensor devices that
are illustrative of a variety of alternative sensor embodiments are
disclosed in a U.S. application entitled "Method and Apparatus for
Electrochemical Analysis," filed concurrently herewith and
incorporated by reference in its entirety. In addition, one skilled
in the art will appreciate that while the illustrated sensors use
vents to control the flow of fluid between chambers, other fluid
directing embodiments are also contemplated. For example, a
physical barrier between the reaction chamber and the detection
chamber could be removed or opened to permit the flow of fluid
between chambers. The sensors described herein could also include
pumping elements to move fluids through the device.
[0039] The immunosensor of the present invention includes
electrodes 52, 152 and 54, 154 as described above. In certain
embodiments, an electrode configuration other than the opposing
relationship illustrated in the FIGS. may be used, for example, a
side-by-side relationship, or an offset relationship. The
electrodes may be identical or substantially similar in size, or
may be of different sizes and/or different shapes. The electrodes
may comprise the same conductive material, or different materials.
Other variations in electrode configuration, spacing, and
construction or fabrication will be apparent to those of skill in
the art.
[0040] In one embodiment, the electrodes are mounted in a parallel
opposing relationship at a distance of less than or equal to 500,
450, 400, 350, 300, 250, or 200 microns, and more preferably from
about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 microns to about 75,
100, 125, 150, or 175 microns. In certain embodiments, however, it
may be preferred that the electrode spacing is greater than 500
microns, for example, 600, 700, 800, 900, or 1000 microns, or even
greater than 1, 2, 3, 4, or 5 millimeters.
[0041] At least one of the electrodes can be a sensing electrode,
i.e., an electrode sensitive to the amount of reduced redox agent
in the antioxidant case or oxidized redox agent in the oxidant
case. In the case of a potentiometric sensor wherein the potential
of the sensing electrode is indicative of the level of analyte
present, a second electrode, acting as reference electrode is
present which acts to provide a reference potential. In the case of
an amperometric sensor wherein the sensing electrode current is
indicative of the level of analyte in the sample, at least one
other electrode is present which functions as a counter electrode
to complete the electrical circuit. This second electrode may also
function as a reference electrode. Alternatively, an additional
electrode (not shown) may perform the function of a reference
electrode.
[0042] In one aspect, the electrically conductive film defining
electrodes 52, 152, 54, 154 can be adhered to a surface the
immunosensor by means of an adhesive. Suitable adhesives include,
for example, heat activated adhesives, pressure sensitive
adhesives, heat cured adhesives, chemically cured adhesives, hot
melt adhesives, hot flow adhesives, and the like. In an alternative
aspect, the electrically conductive film is prepared by coating
(e.g., by sputter coating or screen printing) a sheet of
electrically resistive material with a suitable electrically
conductive material, for example, platinum, palladium, carbon,
indium oxide; tin oxide, mixed indium/tin oxides, gold, silver,
iridium, mixtures thereof, and the like. Materials suitable for use
as the electrodes should be compatible with the reagents present in
the sensor 20, 120. Suitable electrically resistive materials
include, for example, polyesters, polystyrenes, polycarbonates,
polyolefins, mixtures thereof, and the like.
[0043] Reagents for use in the immunosensor, e.g., immobilized
antibody/antigen, probe-linked antigen/antibody, buffer, mediator,
enzyme substrate, and the like, may be supported on the walls the
reaction chamber 22, 122 or on an independent support contained
within chambers, within a matrix, or may be self supporting. If the
reagents are to be supported on the chamber walls or the
electrodes, the chemicals can be applied by use of printing
techniques well known in the art, e.g., ink jet printing, screen
printing, lithography, and the like. In an alternative embodiment,
a solution containing the reagent is applied to a surface within a
chamber and allowed to dry.
[0044] In another embodiment of the immunosensor described herein,
immunological species and/or electrochemical reagents can be
supported on and/or contained within one or more independent
supports which are placed into the sensor. Suitable independent
supports include, but are not limited to, mesh materials, nonwoven
sheet materials, fibrous filling materials, macroporous membranes,
sintered powders, and/or beads. The advantages of independent
supports include an increased surface area, thus allowing more
immobilized binding sites and probe conjugate to be included in the
reaction chamber 22, 122. In one embodiment, an immobilized
antibody and/or probe conjugate are dried onto support materials,
which are then placed into the reaction chamber. Alternatively,
either the immobilized binding site or probe conjugate is
incorporated onto a support material and the other component is
supported on the reaction chamber wall. In yet another embodiment,
the walls of the reaction chamber are porous, with the immobilized
binding site and/or probe conjugate incorporated therein. This can
be accomplished by using a macroporous membrane to form the
reaction chamber wall and compressing the membrane around the
reaction chamber to prevent leakage of sample out of the desired
area.
[0045] Suitable independent supports include material such as mesh
materials, nonwoven sheet materials, and fibrous fill materials
include, polyolefins, polyesters, nylons, cellulose, polystyrenes,
polycarbonates, polysulfones, mixtures thereof, and the like.
Suitable macroporous membranes may be prepared from polymeric
materials including polysulfones, polyvinylidene difluorides,
nylons, cellulose acetates, polymethacrylates, polyacrylates,
mixtures thereof, and the like.
[0046] In one embodiment, the immobilized binding site and/or probe
conjugate is supported on beads. Such beads may comprise a
polymeric material, e.g., agarose, polystyrene, polymethacrylate,
polymethylmethacrylate, optionally encasing a magnetic material
(such as gamma Fe.sub.2O.sub.3 and Fe.sub.3O.sub.4). The bead
material is selected such that suitable support for the antibody is
provided. Suitable beads may include those marketed as
DYNABEADS.RTM. by Dynal Biotech of Oslo, Norway. Optionally, a
magnet may be included to hold the magnetic beads in the reaction
chamber and to stop them from moving to the detection chamber. For
example, the immobilized biding site can be positioned on magnetic
beads within the reaction chamber.
[0047] Using the Sensor to Determine the Presence or Absence of an
Antigen
[0048] As discussed above, sensor 20, 120 can include an
immobilized binding site and a probe conjugate. While the following
description is made with respect to sensor 20 of FIG. 2, it will be
apparent that it applies to sensor 120 as well.
[0049] In one embodiment, an immobilized binding site 44 is an
antibody to the antigen to be detected and the probe conjugate 50
is an enzyme linked to the antigen to be detected or a
pseudo-antigen of the antigen to be detected.
[0050] The antibodies 44 can be adsorbed or otherwise immobilized
therein such that they do not move from the reaction chamber during
a test. Optionally, after application of the antibodies 44 to the
internal surface the reaction chamber, an agent designed to prevent
non-specific binding of proteins to this surface can be applied
(not shown). An example of such an agent well known in the art is
bovine serum albumin (BSA). A nonionic surfactant may also be used
as such an agent, e.g., TRITON X100 manufactured by Rohm & Haas
of Philadelphia, Pa., or TWEEN manufactured by ICI Americas of
Wilmington, Del. Preferably, the nonionic surfactant selected does
not denature proteins.
[0051] Spaced apart from the antibodies is probe conjugate 50
(enzyme-linked antigen). Examples of suitable enzymes for use with
probe conjugate 50 include, but are not limited to, glucose oxidase
and glucose dehydrogenase. The enzyme-linked antigen 50 can be
deposited within the reaction chamber in such a way that it can be
liberated into the sample when wetted by the sample. For example,
the enzyme-linked antigen 50 can be dried on a surface within the
reaction chamber, such that only a weak bond between the
enzyme-linked antigen 50 and the reaction chamber exists. In one
aspect, the rate of dissolution of the enzyme-linked antigen 50 is
chosen such that probe conjugate will dissolve in a sample during
the time taken for the sample to fill the reaction chamber. In this
manner, the enzyme-linked antigen 50 can be evenly distributed
throughout the area of the reaction chamber after filling.
[0052] In one aspect, the relative amounts of enzyme-linked antigen
50 and antibody 44 can be chosen such that there is an excess of
antibody 44 over enzyme-linked antigen 50. In one aspect, an excess
is defined to be such that the excess is small when compared to the
number of antigen molecules to be detected in the sample.
[0053] Thus, when sample fills the reaction chamber the
enzyme-linked antigen 50 mixes with the sample. Sufficient time is
then allowed for the enzyme-linked antigen 50 to come into contact
with the antibodies 44. Since there is an excess of antibodies 44,
if no antigen is present in the sample then substantially all, or a
large portion, of the enzyme-linked antigen 50 will bind to the
antibodies 44 and so be effectively immobilized. If target antigen
is present in the sample, the target antigen will contact and bind
to the antibodies 44, blocking at least some of the enzyme-linked
antigen 50 from binding with the antibodies 44. So, when the target
antigen is present in the sample, then at the end of the reaction
step, the enzyme-linked antigen 50 (or at least a measurable
portion thereof) will remain mobile in the sample and can move into
the detection chamber. Conversely, if no target antigen is present
in the sample, then the enzyme-linked antigen 50 will be
immobilized in 48 reaction chamber (or at least a measurable
reduction in the amount remaining mobile in the sample). One
skilled in the art will appreciate that an excess of antibody 44 is
not necessary and that alternatively, the relative amounts of
enzyme-linked antigen 50 and antibody 44 can be equal, or an excess
of enzyme-linked antigen 50 can be present.
[0054] In a second embodiment, the immobilized binding site 44 is
an antibody that can bind to a site on the target antigen and the
probe conjugate 50 includes an enzyme coupled to an antibody that
can bind to bound target antigen. An advantage of using antibodies
for the immobilized binding site and for the probe conjugate is
that the target binding reagents can be intimately mixed and dried
down together during the manufacture of the strip. The distance
that the binding targets must diffuse can be shorter, which
potentially shortens the time required to perform the assay.
[0055] When the sample enters the reaction chamber, the immobilized
binding site in the reaction chamber can bind to one site on the
analyte of interest. The probe conjugate can include a second
antibody that can bind to a second site on the target antigen
attached to the immobilized binding site. When a sample containing
the analyte of interest fills the reaction chamber, the probe
conjugate and the immobilized binding site mix with the sample and
the analyte of interest binds at one site to the probe conjugate
and at a second site to the immobilized binding site. The analyte
therefore forms a link that immobilizes a fraction of the probe
conjugate, where the fraction immobilized can be used to detect the
presence and/or quantify the concentration of the analyte in the
sample. For example, the amount of immobilized probe conjugate can
be quantified by observing the drop in the amount of free probe
conjugate that is transferred to the detection chamber.
[0056] In yet another embodiment, the immobilized binding site 44
can be the target antigen and the probe conjugate 50 can comprise
an enzyme coupled to an antibody that is capable of binding to the
analyte of interest. Preferably the immobilized binding site and
probe conjugate are position separately in the reaction chamber to
prevent or reduce any reaction prior to the introduction of
sample.
[0057] When a sample containing the target antigen fills the
reaction chamber, the target antigen in the sample can bind to the
probe conjugate (antibody), reducing the amount of probe conjugate
that binds to the immobilized binding site (antigen). The presence
or absence of the target antigen therefore changes the amount of
probe conjugate bound in the reaction chamber. The fraction
immobilized can be used to detect the presence and/or quantify the
concentration of the analyte in the sample. For example, the amount
of immobilized probe conjugate can be quantified by observing the
drop in the amount of free probe conjugate that is transferred to
the detection chamber.
[0058] In one aspect, the immobilized binding sites and/or the
probe conjugate are bound to beads. For example, the immobilized
binding sites (antigen) can be positioned on beads that are dried
on one surface of the reaction chamber and the probe conjugate
(antibody) can be dried on another surface of the reaction chamber.
The beads can be magnetic beads that are prevented from leaving the
reaction chamber by means of a magnetic field. When sample fills
the reaction chamber, antigen in the sample binds to the antibody
of the probe conjugate and prevents the immobilized binding site
(antigen) from the binding with the probe conjugate, thus leaving
the probe conjugate free to be transferred to the detection
chamber.
[0059] As mentioned above, beads can have characteristics such that
they will remain in the reaction chamber when the sample transfers
to the detection chamber. For example, the para-magnetic beads can
be aligned with field lines of an applied magnetic field such that
they are held by the field and thus prevented from being
transferred with the sample to detection chamber. The magnetic
field can be applied by any suitable device such as an
electromagnet or, in an alternative embodiment when it is desired
to minimize power consumption, the magnetic field can be applied by
a permanent magnet. In one embodiment the magnet or magnets could
be placed so that they are closer to the location in the reaction
chamber of the probe conjugate and further from the location in the
reaction chamber of the para-magnetic beads. With this arrangement,
the beads will tend to move towards, and mix with, the probe
conjugate under the influence of the magnetic field. Once the beads
have moved to and mixed with the probe conjugate, the applied
magnetic field will tend to prevent the beads from moving to
locations with a lower concentration of magnetic field lines, thus
the beads will be immobilized in the reaction chamber by the
magnetic field.
[0060] Regardless of the configuration of the immobilized binding
site and the probe conjugate, after the sample reactions within the
reaction chamber, the reacted sample is moved to the detection
chamber. This can occur at a predetermined time after the sample is
introduced into the reaction chamber. For example, the
predetermined time can be set such that there is sufficient time
for substantially all of the probe conjugate to bind. In one
aspect, the residence time of the sample in the reaction chamber
can be calculated by a user manually. Alternatively, the residence
time can be calculated electronically by a meter in electrical
contact with the sensor.
[0061] In one embodiment, the residence time of the sample in the
reaction chamber is monitored via electrodes. For example, in the
sensor of FIGS. 1 and 2 when sample fills the reaction chamber 22,
a small portion of the detection chamber 28 at its opening 38 into
the reaction chamber 22 will be wet by sample. The electrodes 52
and 54 can be placed in the detection chamber 28, such that at
least a portion of each electrode 52 and 54 is contacted by the
sample during the filling of the reaction chamber 22, such that the
presence of the sample will bridge the electrodes 52 and 54 and
create an electrical signal which can be used to trigger the timing
device. In another embodiment, illustrated in the sensor of FIGS. 3
and 4, a separate electrically contact (contact 103), used in
conjunction with the lower electrode and contact area 101, can
detect the presence of sample in the reaction chamber.
[0062] A predetermined time after the sample has entered the
reaction chamber, the immunological reaction phase of the test is
deemed to be completed. The vent 30, 130 can then be opened to the
atmosphere. For example, a solenoid activated needle in the meter
may be used to pierce layer the vent. The piercing can be
automatically performed by the meter or manually by the user, e.g.,
the user inserts a needle through the layer(s) covering the
vent.
[0063] Optionally disposed in the detection chamber 28, 128 are
dried reagents 64 comprising an enzyme substrate and a mediator,
capable of reacting with the enzyme part of the probe conjugate to
produce a detectable signal. The enzyme substrate and mediator, if
present, can be of sufficient amount such that the rate of reaction
of any enzyme present with the enzyme substrate is determined by
the amount of enzyme present. For instance, if the enzyme were
glucose oxidase or glucose dehydrogenase, a suitable enzyme
mediator and an enzyme substrate such as glucose (if not already
present in the sample) would be disposed into detection chamber 28,
128. In one alternative embodiment sufficient glucose would be
disposed into the detection chamber 28, 128 such that any
variations in the level of glucose in the incoming sample did not
significantly alter the enzyme reaction rate. Buffer may also be
included to help adjust the pH of the sample in detection chamber
28, 128. In one embodiment ferricyanide is a suitable mediator.
Other suitable mediators include dichlorophenolindophenol and
complexes between transition metals and nitrogen-containing
heteroatomic species. Additionally, a second mediator such as
phenazine ethosulphate, and/or 2,3
dimethoxy-5-methy-p-benzoquinone, which promotes a more efficient
transfer of electrons from the enzyme to the ferricicyanide species
can be added. The enzyme substrate, mediator, second mediator, and
buffer reagents 64 can be present in sufficient quantities such
that the rate of reaction of the enzyme with the enzyme substrate
is limited by the concentration of the enzyme present.
[0064] When the detection chamber 28, 128 is filled, the reagents
64 dissolve into the sample. The enzyme component of the probe
conjugate reacts with the enzyme substrate and the mediator to
produce reduced mediator. This reduced mediator is
electrochemically oxidized at an electrode acting as an anode in
the detection chamber 28, 128 to produce an electrical current. In
one embodiment, the rate of change of this current with time is
used as an indicator of the presence and amount of enzyme that is
present in the reacted sample. If the rate of change of current is
less than (or more than) a predetermined threshold value, then it
indicates that no significant amount (or a significant amount) of
probe conjugate 50 is present in the reacted sample, indicating the
presence (or lack) of antigen present in the original sample.
Conversely, the rate of change of a current more than (or less
than) a predetermined threshold value can be used to indicate the
lack (or presence) of an antigen in the sample. In one embodiment,
the rate of change of the current is used to give a measure of the
relative amount of antigen initially present in the sample. For
example, the rate of change of current can be used to determine
probe conjugate concentration, which can be correlated to the
concentration of the antigen in the sample.
[0065] Use of Melittin as a Probe.
[0066] In one embodiment, a probe-linked antigen comprising an
antigen-melittin complex can be dried on a wall of the reaction
chamber, as described above. The detection chamber can contain a
mediator comprising ferrocyanide in liposomes or lipid vesicles. If
the antigen-melittin complex reaches the liposomes, they will burst
and release the ferrocyanide. This leads to a rapid amplification
of the signal, i.e., a small amount of free antigen competes with
the antigen-melittin complex for binding sites on the antibodies
and results in a large concentration of ferrocyanide.
[0067] Use of Horse Radish Peroxidase and Alkaline Phosphatase in
Electrochemical Assays.
[0068] Conventional ELISAs use horse radish peroxidase (HRP) or
alkaline phosphatase (AP) as the enzymes in a calorimetric assay.
However, substrates have been developed which allow both these
enzymes to be used in an electrochemical assay. In this embodiment,
AP can be used with p-aminophenyl phosphate and HRP can be used
with tetrathiafulvalene.
[0069] The following non-limiting example is illustrative of the
principles and practice of this invention. Numerous additional
embodiments within the scope and spirit of the invention will
become apparent to those skilled in the art.
EXAMPLE
[0070] An exemplary immunosensor assay for human C Reactive protein
in whole blood using magnetic beads coated with Human C reactive
protein was performed.
[0071] C reactive protein (CRP) was covalently attached to 1.5
micron Carboxylated BioMag magnetic beads (Cat no BM570; Bangs
Laboratories, Indianapolis In, USA). 21.9 mg of beads (1 ml) were
washed 4 times with 50 mM MES (Morpholinoethanesulphonic acid
(Sigma-Aldrich, St Louis, Mo. USA) buffer pH 5.2 by incubating with
this buffer and using a magnet to concentrate the beads on the side
of the tube and after 2 min remove the buffer with a transfer
pipette. After the fourth wash, the beads were suspended in a final
volume of 0.34 ml 50 mM MES. 40 ul of 100 mg/ml EDAC (Sigma, St
Louis, Mo. USA) was added and after 5 minutes 450 ug of CRP in
Phosphate buffered saline (Hytest, Turku Finland) was added. Beads
were allowed to incubate for a further 30 min at room temperature.
Unbound CRP was removed by using a magnet to concentrate the beads
as described above. The beads were then blocked by incubating for
30 min in a buffer containing 20 mM Tris
(2-amino-2-hydroxymethyl)-1,3 propandiol), 0.15 M Sodium Chloride
pH 7.4 (TBS) and 1 mg/ml Bovine serum albumin (BSA) (Sigma-Aldrich,
St Louis, Mo. USA) and then washed four times in the same buffer
using magnetic concentration. Beads were stored in TBS/BSA
containing 0.05% sodium azide (Sigma-Aldrich, St Louis, Mo.
USA).
[0072] Glucose Dehydrogenase/Antibody Conjugate.
[0073] Conjugation of Glucose dehydrogenase (Recombinant E. coli
enzyme; Kiikoman Corporation, Chiba, Japan) GDH and monoclonal
antibody 4C28 clone C2 (Hytest, Turku Finland) was accomplished
using the reagent MBS (M-Maleimidobenzoyl-N-Hydroxysuccinimide
ester) based on the method described by O Sullivan et al. (Anal.
Biochem. 100 100-108 1979).
[0074] In this example MBS was reacted with amino groups on GDH and
the MBS-GDH intermediate was purified. Then maleimide groups on the
SMCC-GDH complex were reacted with free sulphydryl groups on the
hinge region of the antibody introduced by reaction with the
reducing reagent cysteamine HCl.
[0075] 1. Reduction of IgG Hinge Region Disulphides
[0076] Six mg of cysteamine HCl (Sigma-Adrich, St Louis, Mo. USA)
was incubated for 90 minuets at 37.degree. C. with 1 ml of a
solution containing 2 mg/ml Mab C2 in a buffer containing 0.1M
Sodium phosphate pH 7.4; 0.15 M NaCl and 2.5 mM EDTA (reaction
buffer). The reaction was terminated by applying the mixture to a
desalting column (PD-10; Amerscham) equilibrated in reaction buffer
and elution continued in the same buffer. One-half milliliter
fractions were collected and the three fractions containing the
most protein were pooled. This material was reacted with the
maleimide activated enzyme as soon as it was pooled. The protein
concentration was determined assuming an absorbance at 280 nm of
1.35 for a 1 mg/ml solution of antibody C2.
[0077] 2. Maleimide Activation of the Enzyme.
[0078] At the same time 2 mg of GDH was dissolved in 1.0 ml of the
reaction buffer and 50 ul of a solution of containing 7.6 mg/ml MBS
(Pierce Rockford Ill. USA) in DMSO was added and allowed to
incubate for 30 min at 37.degree. C. The reaction was terminated by
applying the mixture to a desalting column (PD-10; Amerscham)
equilibrated in the reaction buffer and elution continued in the
same buffer. One-half milliliter fractions were collected and the
three fractions containing the most protein were pooled.
[0079] 3. Conjugation of Antibody to Enzyme
[0080] The reduced IgG and maleimide reacted GDH were mixed
together in the ratio of 1 mg of Ab to 0.9 mg of GDH and incubated
overnight at 4.degree. C. The reaction was terminated by adding 6
mg of cysteamine HCl and allowing to incubate for a further 15 min
at room temperature and then applying 1.5 ml aliquots to separate
desalting columns equilibrated with and continuing the elution in a
buffer containing 20 mM Tris and 0.15 M Sodium Chloride pH 7.4
(TBS). The three 0.5 ml fractions containing the highest
concentration of protein from each column were pooled. Calcium
chloride was added to a final concentration of 1 mM, sodium azide
to a final concentration of 0.1% and PQQ to a final concentration
of 0.05 mg/ml. Conjugate was stored at 4.degree. C. prior to
use.
[0081] Conventional Immunoassay for C Reactive Protein.
[0082] The CRP levels in samples were also determined by
conventional enzyme immunoassay. All incubations were carried out
at room temperature.
[0083] The wells of Immulon 11 microplates were coated with 50 ul
of a 10 ug/ml monoclonal antibody C2 in TBS buffer for 60 min at
room temperature. Unbound antibody was removed by inversion and
tapping the plate and then washing the wells four times with 200 ul
of TBS buffer containing 0.1% TWEEN 20 (Polyoxyethylenesorbitan
monlaurate; Sigma-Adrich, St Louis, Mo. USA).
[0084] Then 50 ul of known standards, or sample containing unknown
amounts of CRP was added, usually diluted 100-1000 fold in
TBS/TWEEN, to each well and allowed to incubate for a further hour.
Unbound antigen was then removed by the washing procedure described
above. Next 50 ul of a 220 ng/ml solution of biotinylated C6
antibody in TBS/TWEEN was added and allowed to incubate for further
60 min. After washing away unbound second antibody, 50 ul of a
1/1000 dilution of neutravidin-horseradish peroxidase was added
(Pierce Rockford Ill. USA) and the reaction allowed to proceed for
a further 15 min. Finally, after washing to remove unbound enzyme,
bound enzyme was detected by the addition of ABTS substrate (Pierce
Rockford Ill. USA).
[0085] Biotinylation of antibody C6 was carried out by the
following method. Two milligrams of monoclonal antibody C6 (Hytest,
Turku Finland) were dissolved 1 ml of 50 mM Sodium bicarbonate and
reacted with 29 ul of 1 mg/ml solution of biotin N
Hydroxysuccinimide ester (Pierce) in Dimethyl sulphoxide (Sigma).
The reaction was allowed to proceed for 30 min with occasional
shaking. The reaction was terminated by applying the mixture to a
desalting column (PD-10; Amerscham) equilibrated in 20 mM Tris
(2-amino-2-hydroxymethyl)-1,3 propandiol), 0.15 M Sodium Chloride
pH 7.4 (TBS) and elution continued in the same buffer. One-half
milliliter fractions were collected and the three fractions
containing the most protein were pooled. The material was stored at
4.degree. C. Protein concentration was determined by absorbance at
280 nm assuming a 1 mg/ml solution of C6 had an absorbance of
1.2.
[0086] Sensor Strips
[0087] Sensor strips were constructed as following: [0088] 1) The
electrode webs consisted of 178 um thick Melinex which was sputter
coated with a layer of gold. The surface resistance of the gold
coating was 8-12 ohms/sq. [0089] 2) The electrode was coated with a
solution of 0.3 mM 2-mercaptoethanesulphonic acid for 20 seconds
and then dried with a jet of air. This procedure keeps the
electrode hydrophilic and reduces fouling by air-borne hydrocarbons
and other contaminants. [0090] 3) A web process was used to dry
stripes of chemistry (e.g., reagents described above) onto the
electrode. The web was transported past a fixed razor blade which
placed a scratch on the surface and helped to define the area of
the working electrode. The web was then transported past two
blunt-tipped stainless steel pipetting needles linked to a syringe
pump which deposited: [0091] the electrochemical reagent
(ferricyanide, glucose, etc.) so that it overlapped the scratch,
and [0092] the antibody-enzyme conjugate on the other side of the
scratch about 1-2 mm away from the electrochemical reagent. [0093]
4) The chemistry stripes were dried with infrared dryers and hot
air at 50 degrees C. [0094] 5) The reaction and detection chamber
shapes were kiss-cut into the spacer using a rotary crush-cut tool.
[0095] 6) The spacer was laminated onto the electrode in such a way
that the antibody-enzyme conjugate was in the reaction chamber and
the electrochemical reagent was in the detection chamber. [0096] 7)
The filling chamber was punched into the bi-laminate using a
male/female die set. [0097] 8) Antigen bound to paramagnetic beads
was striped onto a separate electrode film. [0098] 9) The electrode
with the paramagnetic beads was then bonded to the bi-laminate from
step (6) in such a way that the antigen-bead stripe was opposite
the antibody-enzyme conjugate in the reaction chamber. [0099] 10)
The vent hole in each sensor was punched into the tri-laminate
using a male/female die set. [0100] 11) The vent holes and the open
side of the filling chamber were covered with "magic tape" (3M).
[0101] 12) The tri-laminate web was singulated to yield working
sensors.
[0102] Electrochemical Detection of GDH.
[0103] The solution contained 5 mg/ml 2,3 Dimethoxy-5-methyl 1,4
benzoquinone (Adrich, Wis. USA) 326 mg/ml potassium ferricyanide
400 mM glucose in a buffer containing 0.26 mg/ml citraconic acid
(Sigma) and 13.3 mg/ml Di potassium citraconate.
[0104] Conjugate
[0105] Solution contained 400 ug/ml GDH was diluted to 100 ug/ml in
a solution containing 1 mM Calcium chloride, 10 mg/ml BSA 0.26
mg/ml citraconic acid, 13.3 mg/ml Di potassium citraconate, and 10
mg/ml sucrose.
[0106] Magnetic Beads
[0107] Solution contained 5 mg/ml CRP coated beads, 100 mg/ml
sucrose, 1 mM Calcium chloride, 0.26 mg/ml citraconic acid, and
13.3 mg/ml Di potassium citraconate
[0108] Sample Preparation
[0109] Normal heparainized whole blood with an Hemocrit of 42% and
plasma concentration of 1 ug/ml CRP was used for the following
experiment. (CRP blood), To 100 ul of whole blood 10 ul of a
solution of 2.5 mg/ml CRP in phosphate buffed saline was added. 10
ul of phosphate buffed saline was added to another 100 ul sample as
a control.
[0110] Test Procedure
[0111] Approximately 5 ul of blood was added to the filling chamber
(feature 107 of FIG. 3). The blood flowed to fill filling chamber
107 and reaction chamber 122 and stopped near the entrance to
detection chamber 128. The blood dissolved the conjugate from the
wall of reaction chamber 122 and allowed interaction with the
magnetic beads which were drawn to the bottom of reaction chamber
122 by the presence of a magnet under the reaction chamber 122. In
the absence of added CRP the majority of conjugate will be able to
bind the magnetic beads.
[0112] After incubation for 40 seconds the vent hole 130 in FIG. 3
was punctured. This allowed blood together with any unbound GDH
conjugate to flow past the scratch line (106) into the detection
chamber where measurement of the electrical current flowing between
the electrodes in the detection chamber was initiated. The current
generated by the presence of GDH in the detection chamber was
measured over the next 45 s. The results below are for six
replicate samples of the control blood or blood containing 250
ug/ml CRP. They show the current in .mu.A at 5 seconds and 45
seconds after the detection chamber 128 was filled. The difference
in the current between 5 and 45 seconds was used as a measure of
the CRP concentration in the sample. TABLE-US-00001 Control sample
With 250 ug/ml CRP added 5 sec 45 sec Difference 5 sec 45 sec
Difference 19.01 27.72 8.71 24.26 40.8 16.54 16.99 26.45 9.46 20.32
37.25 16.93 20.05 28.41 8.36 26.39 47.25 20.86 18.81 31.1 12.29
25.12 50.44 25.32 16.75 24.0 7.05 20.41 39.09 18.68 22.94 34.22
11.28 17.93 35.77 17.84 Avg. 9.525 19.36 Std dev. 1.77 1.55
[0113] As shown in the table above, there was a significant
difference in the rate of change of current between the control
sample and the sample to which 250 ug/ml CRP was added. The
immunosensor was thus able to detect the presence of CRP within the
sample.
[0114] One skilled in the art will appreciate further features and
advantages of the invention based on the above-described
embodiments. Accordingly, the invention is not to be limited by
what has been particularly shown and described, except as indicated
by the appended claims. All publications and references cited
herein are expressly incorporated herein by reference in their
entirety.
* * * * *