U.S. patent application number 10/023797 was filed with the patent office on 2002-05-09 for method for continuous immunoassay monitoring.
Invention is credited to Gindilis, Andrei L..
Application Number | 20020055127 10/023797 |
Document ID | / |
Family ID | 22527939 |
Filed Date | 2002-05-09 |
United States Patent
Application |
20020055127 |
Kind Code |
A1 |
Gindilis, Andrei L. |
May 9, 2002 |
Method for continuous Immunoassay monitoring
Abstract
The present invention relates to assays that employ an enzyme
label or tag that acts on a substrate by obtaining electrons from
an electrode (electrocatalysis) and an apparatus for use in such
assays. In particular the present invention provides an assay and
apparatus for multiple or continuous monitoring of the amount of
analyte in a sample or sample source without requiring regeneration
of the measuring electrode or its associated reagents.
Inventors: |
Gindilis, Andrei L.;
(Mukilteo, WA) |
Correspondence
Address: |
CARELLA, BYRNE, BAIN, GILFILLAN, CECCHI,
STEWART & OLSTEIN
6 BECKER FARM ROAD
ROSELAND
NJ
07068
US
|
Family ID: |
22527939 |
Appl. No.: |
10/023797 |
Filed: |
December 17, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10023797 |
Dec 17, 2001 |
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09148900 |
Sep 8, 1998 |
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Current U.S.
Class: |
435/7.9 ;
205/777.5 |
Current CPC
Class: |
Y10S 435/817 20130101;
Y10S 436/806 20130101; Y10S 436/807 20130101; G01N 2333/62
20130101; G01N 33/5438 20130101; G01N 33/535 20130101; C12Q 1/001
20130101 |
Class at
Publication: |
435/7.9 ;
205/777.5 |
International
Class: |
G01N 033/53; G01N
033/542; G01F 001/64 |
Claims
What is claimed is:
1. A method for continuous determination of a target analyte in a
sample comprising the steps of: (a) contacting an analyte with an
immunosensory apparatus comprising a working electrode having a
binder on its surface and a reference electrode and wherein said
working electrode requires no regeneration between consecutive
measurements of the analyte; (b) determining a change in the
working electrode potential, and (c) comparing the potentiometric
response of the working electrode with the reference electrode
wherein a difference indicates the presence of analyte in said
sample and said difference is proportional to the concentration of
analyte, and wherein said measurement can be performed with a
single working electrode, thereby determining the concentration of
said analyte in the sample.
2. The method of claim 1 wherein said analyte is selected from the
group consisting of an antigen, a hapten and an antibody.
3. The method of claim 1 wherein said labeled detection compound is
selected from the group consisting of an antigen, a hapten and an
antibody.
4. The method of claim 1 wherein said labeled detection compound is
the same as the analyte.
5. The method of claim 1 wherein said labeled detection compound is
a binder for the analyte.
6. The method of claim 1 wherein the binder is a binder for both
the analyte and the labeled detection compound.
7. The method of claim 1 wherein the analyte binds reversibly to
the binder.
8. The method of claim 1 wherein said electrocatalytic enzyme an
oxidoreductase.
9. The method of claim 1 wherein said electrocatalytic enzyme is a
member selected from the group consisting of laccase, lactate
dehydrogenase, horseradish peroxidase, cytochrome c peroxidase, a
fungal peroxidase, lactoperoxidase, microperoxidase,
chloroperoxidase, hydrogenase, D-fructose dehydrogenase,
methylamine dehydrogenase, flavocytochrome c552, succinate
dehydrogenase, fumarate reductase, alcohol dehydrogenase,
D-gluconate dehydrogenase, cellobiose dehydrogenase and ascorbate
oxidase.
10. The method of claim 1 wherein said electrocatalytic enzyme is
laccase.
11. The method of claim 1 wherein the diffusion medium is a liquid
or a gel.
12. The method of claim 1 wherein said diffusion medium contains a
second substrate for the electrocatalytic enzyme and the analyte
binds to one of either the binder on said working electrode or the
detection compound.
13. The method of claim 12, wherein said second substrate is
oxygen.
14. A method for determining a target analyte in a sample
comprising the steps of: (a) contacting an analyte with an
immunosensory apparatus, comprising a diffusion membrane, a
reference electrode and a working electrode, the latter having on
its surface a binder with a labeled detection compound attached
thereto, wherein said electrodes and said membrane are separated by
a chamber containing a diffusion medium that contains a substrate
of an electrocatalytic enzyme and wherein said electrodes are
separated from the source of said analyte by said membrane, under
conditions promoting diffusion of said analyte through said
membrane to contact said electrodes; (b) allowing the analyte to
displace the labeled detection compound from said binder whereupon
said analyte becomes bound to the binder on the surface of the
working electrode in the case where the detection compound is the
same as the analyte or the analyte becomes bound to the detection
compound in the case where the labeled detection compound is a
binder for the analyte, and wherein said detection compound is
bound to an electrocatalytic enzyme such that in the presence of
the target analyte the working electrode provides an electron to
said electrocatalytic enzyme which provides an electron to a
substrate of said enzyme (c) determining a change in the working
electrode potential, and (d) comparing the potentiometric response
of the working electrode with the reference electrode wherein a
difference indicates the presence of analyte in said sample and is
proportional to the concentration of analyte, thereby determining
the concentration of analyte in the sample.
15. The method of claim 14 wherein electrode regeneration is not
required between successive determinations of said analyte.
16. The method of claim 14 wherein regeneration of the working
electrode and other reagents is not required between successive
determinations of said analyte.
17. A method for intermittently or continuously conducting
immunoassay measurements wherein a plurality of different
measurements for an analyte are available for a single electrode
without requiring regeneration of said electrode and other
reagents, wherein said method is for determining a target analyte
based on displacement activity of the target analyte and a
potentiometric mode, said method comprising the following steps:
(a) immersing of the intermediate and/or continuous
bioelectrocatalysis immunoassay sensing element of claim 8 in a
assay medium containing the target analyte, (b) allowing the target
analyte of the assay medium to diffuse through the diffusion
membrane of the sensing device and travel to the surface of the
working electrode, (c) permitting an immuno-equilibrium to be
established within the sensing device with respect to the amount of
target analyte present in the assay medium due to displacement of
some or all of a labelled detection compound from the binder on the
surface of the electrode by the target analyte which target analyte
becomes bound to the binder on the surface of the sensing element
in the case where the detection compound is the same as the analyte
or the target analyte becomes bound to the detection compound in
the case where the detection compound is a binder for the analyte,
(d) measuring at least one shift of the electrode potential caused
by the displacement of some or all of the labeled detection
compound from the electrode's surface and the resulting
diminishment or absence of electrocatalytic properties caused by
the label of detection compound, (e) determining the potentiometric
sensor response which is proportional to the degree of displacement
of the labeled detection compound from the binder on the surface of
the working electrode caused by competitive binding of the target
analyte, and (f) determining the concentration of the target
analyte in the external media from the potentiometric sensor
response as compared with the control electrochemical reference
electrode.
Description
[0001] The present invention relates to assays that employ an
enzyme label or tag that acts on a substrate by obtaining electrons
from an electrode (electrocatalysis) and an apparatus for use in
such assays.
BACKGROUND OF THE INVENTION
[0002] Immunoassay techniques are based on the ability of
antibodies to form complexes with the corresponding antigens or
haptens. This property of highly specific molecular recognition of
antigens by antibodies leads to high selectivity of assays based on
immune principles. The high affinity of antigen-antibody
interactions results in great sensitivity of immunoassay methods.
The use of a label or indicator to verify that an antigen/antibody
interaction has occurred is the basis for immunoassay methods.
[0003] Immunoassay techniques have been used mainly in clinical
analyses and medical diagnostics. However, immunoassay applications
in other areas such as environmental control, food quality control,
etc. are expanding. Certain limitations in assaying techniques due
to existing procedures have limited somewhat the expansion into
such other areas.
[0004] In this respect, during the last few years a significant
number of publications have dealt with non-conventional
(alternative) immunoassay techniques designed to expand the
accuracy or applicability of immunoassays. In most cases the
development of alternative immunoassay techniques aims at
improvements in performance of conventional immunoanalysis. Often
such improvement attempts are directed to decreasing analysis
times, increasing assay sensitivity, and simplifying and automating
assay procedures.
[0005] For example, the utilization of enzymes able to catalyze
electrochemical reactions by direct (mediatorless) mechanism
(bioelectrocatalysis) would allow for the detection of
immuno-interactions in real time. Such applications of
bioelectrocatalysis in the development of immunosensors are based
on the self-assembling or displacement of molecule/label complexes
or "molecular transducers" on the surface of an electrode that has
been modified by immunospecies that bind the complex. Ordinarily
these immunospecies would be complimentary to the immunoconjugate
which includes the electrocatalytically active enzyme-label.
[0006] Antigen immobilized on the electrode surface interacts with
the enzyme-labeled antibody which results in the attachment of the
enzyme to the electrode surface. Attachment of the electrocatalytic
active enzyme on the electrode surface initiates, in the presence
of a substrate, an electrocatalytic reaction. Therefore, the
formation of an antigen-labeled antibody complex on the electrode
surface is accompanied by an assembling of the molecular
transducing layer. The rate of electron transfer can be limited by
the efficiency of electrical connection between the enzyme-label
and the electrode surface, which is already modified by the
immobilized immunospecies.
[0007] A potentiometric immunosensor based on mediatorless
bioelectrocatalysis has been utilized which employed laccase enzyme
as an electrocatalyst-label. The electrocatalytic property of the
enzyme in the reaction of oxygen electroreduction (reaction 1)
allowed the detection of the biospecific interaction of a
laccase-labeled receptor, or antibody, with a ligand modified
electrode. Formation of a complex between the laccase labeled
antibody and antigen on the electrode surface results in a
considerable shift in electrode potential due to the catalytic
reduction of over voltage. Analysis was performed in a competitive
scheme, and a single measurement was made with 20 minutes. Such a
potentiometric immunoassay does not require an electrochemically
active mediator. The reaction substrates were atmospheric oxygen
and electrons that were transferred directly from the electrode to
the oxygen molecule via the active site of the enzyme. Insulin was
used as a model analyte.
[0008] In the above immunoassay sensor, the electron which is the
"second substrate" of enzymatic reaction can be captured by the
enzyme-label only from the electrode surface. Therefore, only
molecules intimately attached to the electrode surface generate
electrochemical signal. The rate of attachment of electrocatalyst
molecules to the electrode surface is proportional to the rate of
formation of the immuno-complex on the electrode surface. The rate
of attachment of electrocatalyst molecules to the electrode surface
is proportional to the rate of formation of the immuno-complex on
the electrode surface. The rate of immunointeraction on the
electrode surface can be directly monitored by amperometric or
potentiometric mode.
[0009] However, assays based on mediatorless bioelectrocatalysis
are limited in that primarily one of the two assay procedures set
forth below are utilized and only a single assay measurement may be
taken before the electrode is regenerated or replaced by a new
electrode. The two competitive assay procedures are:
[0010] (a) Competitive Immunoassay With an Initial Label-free
Electrode--An electrode having no attached analyte/enzyme label is
utilized as a starting point and a measured amount of analyte media
along with a measured quantity of analyte/enzyme label are assayed
by a competitive binding assay procedure. After maximum association
with the electrode has occurred the amperometric or potentiometric
measure result is compared to that of an electrode having 100%
analyte/enzyme label associated. The difference in measurements
corresponds proportionally to the amount of analyte in the media
being assayed. and
[0011] (b) Displacement Immunoassay With an Initial Label-loaded
Electrode--An electrode having the maximum amount of attached
analyte/enzyme label (a fully loaded electrode) is utilized as a
starting point and a measured amount of analyte media is assayed by
a competitive binding assay procedure. After maximum displacement
of the analyte/enzyme label from the electrode by the analyte of
the media has occurred the amperometric or potentiometric measure
result is compared to that of the initial fully loaded electrode
(having 100% analyte/enzyme label associated). The difference in
measurements corresponds proportionally to the amount of analyte in
the media being assayed.
[0012] In addition to the laccase enzyme label, the potentiometric
immunosensor employing peroxidase as an electrocatalyst-label has
also been developed. The basic principle is the same as for the
laccase based immunosensor. The electrode surface is modified by an
immobilized antigen (rabbit IgG). The peroxidase-antibody conjugate
associates with the antigen on the electrode surface. Once added to
the media, and on reaching the electrode surface, the
antibody-conjugated peroxidase starts to catalyze the
electro-reduction of hydrogen peroxide. This results in an increase
(anodic shift) in the electrode potential.
[0013] Both the laccase and peroxidase label immunosensors based on
bio-electrocatalytic detection (as discussed above) allow direct
detection of immunointeraction in real time. However, these sensors
must be regenerated or replaced (e.g., disposable sensors) after
each measurement. Accordingly, such immunoassay procedures do not
allow continuous monitoring of the analyte. In addition, such
procedures are a multi-stage process that result in a general
complexity of analysis and require a highly qualified technician to
conduct the assay.
[0014] Accordingly, there is a need for immunoassay procedures that
can be continuous, particularly automatable procedures or
procedures that do not require highly qualified technicians to
conduct the assay.
SUMMARY OF THE INVENTION
[0015] An object of the present invention is to provide an improved
bioelectrocatalysis immunoassay apparatus for detecting an analyte,
the apparatus comprising a sensing device with an electrode,
wherein the sensing device has the ability to monitor changes in
the amount of analyte in a liquid without requiring regeneration or
replacement of the electrode or of the reagents used in the assay.
Preferably, the sensing device of the apparatus is capable of
multiple intermittent and/or continuous immunoassay measurements of
the same analyte without a requirement for regeneration or
replacement of the electrode or other reagents.
[0016] In one aspect the apparatus is for detecting an analyte that
is an antigen, antibody or hapten by use of a labeled detection
compound that is labelled with an electrocatalytic enzyme, wherein
the labeled detection compound is either the analyte or the binder
for the analyte, and wherein the apparatus provides an electrode to
which is permanently affixed (i) a binder for the analyte in the
case wherein the labeled detection compound is a labeled analyte or
(ii) the analyte in the case wherein the labeled detection compound
is a labeled binder for the analyte. If such detection compound
becomes closely associated with the electrode of the apparatus, the
electrocatalytic enzyme label or tag will interact with the
electrode to cause a detectable electrical change for the
electrode. In one embodiment the analyte is the same antigen,
antibody or hapten as the detection compound (wherein the detection
compound is labeled with an electrocatalytic enzyme or tag) and
some of the fixed amount of the labelled detection compound complex
that is detectable by the electrode due to bioelectrocatalysis
competes with the analyte for binding to the binder of the
electrode. Alternatively, the binder on the electrode and the
analyte are the same antigen, antibody or hapten and compete for
binding to the detection compound that is labelled with the
electrocatalytic enzyme.
[0017] In accordance with the above apparatus (depending upon
whether the analyte is the same as the binder or the same as the
detection compound) the analyte will bind to either the binder or
to the detection compound. In either event, the amount of the
detection compound that attaches to the binder is inversely
proportional to the concentration of the analyte in the sample
being analyzed. The amount of the detection compound which is bound
to the binder of the electrode is indirectly measurable by
bioelectrocatalysis and the amount of analyte which is present in
the sample is therefore detectable by the amount of
bioelectrocatalysis occurring at the electrode's surface. In each
of the above cases (i.e., (i) when the binder and analyte are the
same and (2) when the binder and the detection compound are the
same, the electrode only transfers an electron to the label as a
substrate when the labeled detection compound is attached to the
binder affixed to the electrode. Therefore, a shift in potential or
current, can be observed by the amount of labeled detection
compound that binds to the binder on the electrode, which amount of
labeled detection compound bound to the binder on the electrode is
inversely proportional to the amount of analyte in the sample. When
current is measured instead of potential at the electrode, the
current is proportional to the amount of laccase label attached to
the electrode. Measurement of potential is preferred since
amperometric measurement of the electrode signal takes into
consideration the surface area of the electrode and the density of
the laccase label attached to the electrode. Thus, potential
measures are usually more accurate, but sometimes amperometric
measures are more accurate and should be used. The ordinary
practicioner in this field would know when to use a particular
electrode type. Thus, when the terms "potentiometric electrode" and
"potentiometric assay" and the like are used in this application
and claims the word "ampherometric" may be substituted for
"potentiometric".
[0018] A further object of the invention is to provide an improved
bioelectrocatalysis immunoassay sensing device wherein the
electrode is encased by a housing member comprising at least one
porous or semi-porous surface, such as a semipermeable membrane,
that is permeable for an analyte and impermeable to the labelled
detection compound, preferably a detection compound that is
labelled with an electrocatalytic enzyme. In a preferred aspect the
sensing device comprises a particular quantity of the labelled
detection compound which is enclosed within the housing member,
which labelled detection compound may contact the electrode when
the sensing device is placed in a liquid or gaseous medium and the
at least one porous or semi-porous surface is impermeable to the
labelled detection compound.
[0019] A preferred object of the invention is to provide an
apparatus for immunodetermination of target analyte (antigen,
antibody or hapten) in an analyte sample, where said apparatus
comprises
[0020] (a) a sensing device comprising:
[0021] (i) a potentiometric working electrode at least one surface
of which is located within said housing member, wherein said
electrode is connected to a potentiometric measuring circuit and
said electrode has the ability to provide an electron to an enzyme
label which will deliver the electron to a first substrate for the
enzyme label, and said electrode has permanently affixed to at
least one of its surfaces a binder for at least a detection
compound which is labelled with an electrocatalytic enzyme;
[0022] (ii) a housing member comprising at least one surface that
is permeable to an analyte and impermeable to the labelled
detection compound, wherein the detection compound is a member
selected from the group consisting of (a) a binder for the analyte
(in the case where the binder on the electrode is the analyte) and
(b) the analyte (in the case where the binder on the electrode is a
binder for the analyte), and in each case the detection compound is
labelled with an electrolytic enzyme or tag; and
[0023] (iii) an internal media which is located within the housing
member and which is a gel or liquid containing a pre-determined
amount of labelled detection compound as herein above described, or
capable of containing a predetermined amount of the labelled
detection compound; and
[0024] (b) an electrochemical reference electrode connected to a
potentiometric measuring circuit,
[0025] and wherein the internal media of (iii) or the analyte
sample comprises a second substrate for the enzyme label or tag,
such as oxygen. The analyte will either bind to the binder or to
the detection compound.
[0026] In a preferred aspect, the apparatus further comprises at
least one measuring device which is connected directly or
indirectly to the sensing device and/or the reference electrode.
Preferably, the at least one measuring device is a member selected
from a digital voltmeter or other similar measuring device. In one
aspect said measuring device is interfaced with a personal computer
as well as the sensing element and the electrochemical reference
probe. In a preferred aspect, the measuring device is also
connected to a member selected from (i) a signal recorder which is
an X-T recorder, (ii) a microprocessor based data acquisition
system with a digital display, and (iii) a personal computer.
[0027] Additionally, an object of this invention is provide a
sensing device comprising an external housing with at least one
semipermeable surface and having within the housing device at least
one surface of a working electrode comprised of an electrode body
made by electrochemically inert electro-conductive material
modified by a binder immobilized on its surface which will bind to
at least a detection compound which is labelled with an
electrocatalytic enzyme, and the binder may be the same as the
analyte or may be a binder for the analyte. In a preferred aspect,
the binder is a binder for both the analyte and the labeled
detection compound, whereby the binder will reversibly bind
individually to the analyte or to the labelled detection compound
which will each compete to be bound by the binder. Preferably, the
detection compound is labelled with the laccase enzyme which can
use oxygen and electrons from the electrode as substrates.
[0028] Another object of the invention is a sensing element which
comprises (a) a potentiometric working electrode, (b) an external
housing with at least one surface that is a semipermeable surface
(such as a diffusion membrane) through which an analyte may diffuse
and is impermeable to at least one detection compound which is
labelled with an electrolytic enzyme or tag and the detection
compound will bind to either the binder of the electrode, or to
both the binder on the electrode and the analyte of the sample, and
(c) an electrochemical reference electrode. In a preferred aspect
the sensing element further comprises an internal media, which is a
gel or liquid, and a fixed quantity of the labelled detection
compound, or comprises a means for inserting a quantity of labelled
detection compound and/or the internal media comprising a fixed
quantity of the labelled detection compound.
[0029] Another object of the invention is to provide a portable
biosensor capable of continuously detecting a target analyte in a
wide range which operates in a continual potentiometric mode from
which multiple potentiometric measurements are available that
correspond to the amount of analyte present in a given sample being
assayed.
[0030] In yet another aspect, an object of the present invention is
to provide a method for intermittently or continuously conducting
immunoassay measurements wherein a plurality of different
measurements for an analyte are available for a single electrode
without requiring regeneration of said electrode and other
reagents. In particular, such object is accomplished by a method of
determination a target analyte based on displacement activity of
the target analyte and a potentiometric mode comprising the
following steps:
[0031] (a) immersing of the intermediate and/or continuous
bioelectrocatalysis immunoassay sensing element (described above)
in a assay medium containing the target analyte,
[0032] (b) allowing the target analyte of the assay medium to
diffuse through the diffusion membrane of the sensing device and
travel to the surface of the working electrode,
[0033] (c) permitting an immuno-equilibrium to be established
within the sensing device with respect to the amount of target
analyte present in the assay medium due to displacement of some or
all of a labelled detection compound from the binder on the surface
of the electrode by the target analyte which target analyte becomes
bound to the binder on the surface of the sensing element in the
case where the detection compound is the same as the analyte or the
target analyte becomes bound to the detection compound in the case
where the detection compound is a binder for the analyte,
[0034] (d) measuring at least one shift of the electrode potential
caused by the displacement of some or all of the labeled detection
compound from the electrode's surface and the resulting
diminishment or absence of electrocatalytic properties caused by
the label of detection compound,
[0035] (e) determining the potentiometric sensor response which is
proportional to the degree of displacement of the labeled detection
compound from the binder on the surface of the working electrode
caused by competitive binding of the target analyte, and
[0036] (f) determining the concentration of the target analyte in
the external media from the potentiometric sensor response as
compared with the control electrochemical reference electrode.
[0037] In yet further aspect, an object of the present invention is
to provide a method for intermittently or continuously conducting
competitive immunoassay measurements wherein a plurality of
different measurements are available for a single electrode without
requiring regeneration of said electrode. In the situation where
the amount of target analyte changes to become more or less
concentrated in a continuous monitoring process, a competitive
binding immunoassay can be utilized. In particular, such object is
accomplished by a method of determination the variations in amount
of a target analyte based on displacement activity of the
enzyme-labeled detection compound (i.e., labeled analyte) or from a
binder for the analyte and the detection compound which binder is
on the working electrode and corresponding potentiometric responses
to such attachment changes, which method comprises the following
steps:
[0038] (a) immersing of the intermediate and/or continuous
bioelectrocatalysis immunoassay sensing element (described above)
in a assay medium which may vary continuously with respect to the
concentration of the target analyte present,
[0039] (b) allowing the target analyte of the assay medium to
diffuse through the diffusion membrane of the sensing device and
travel to the surface of the working electrode,
[0040] (c) permitting an immuno-equilibrium to be established
within the sensing device with respect to the amount of target
analyte present in the assay medium due to (i) displacement of some
or all of a labeled detection compound (e.g., labeled analyte) that
is reversibly bound to a binder immobilized on the surface of the
electrode by the target analyte which target analyte becomes
reversibly bound to the binder immobilized on the surface of the
sensing element upon displacing the labeled detection compound, or
(ii) displacement of some of the reversibly bound target analyte
from the binder on the surface of the electrode by some of the
fixed amount of the labeled detection compound that is present
within the sensing device due to a lowering of the concentration of
the target analyte which causes a shift in its binding equilibrium
and degree of binding with the binder on the surface of the
electrode,
[0041] (d) measuring at least one shift of the electrode potential
caused by the displacement occurring on the surface of the working
electrode of either the reversibly bound labeled detection compound
or the reversibly bound target analyte and resulting changes in
electrocatalytic properties occurring at the surface of the
electrode,
[0042] (e) determining the potentiometric sensor response which is
proportional to said binding displacement on the surface of the
working electrode,
[0043] (f) determining changes in the concentration of the target
analyte in the external media from changes in the potentiometric
sensor response as compared with the control electrochemical
reference electrode and prior measurements from the working
electrode.
[0044] In accordance with one aspect of the invention, there is
provided an assay procedure wherein a sample containing analyte (a
substance to be determined) placed in contact with an electrode
which supports a binder for the analyte, which binder has bound
thereto a labeled form of the analyte in which the label is an
electrocatalytic enzyme (an enzyme that acts on a substrate by
obtaining electrons from the electrode) and wherein such electrode
is also contacted with a substrate for such enzyme label. The
analyte in the sample displaces some or all of the reversibly bound
labeled analyte from the binder, with the amount of binding
displacement being directly related to the amount of analyte in the
sample. Displacement of labeled analyte results in a change in
voltage between such electrode and a reference electrode and such
change in voltage is a measure of the amount of analyte in the
sample.
[0045] In a preferred embodiment, the electrode having supported
thereon the binder having bound thereto a labeled analyte is
present in a chamber which includes a member or wall which is
permeable to analyte but which is impermeable to the enzyme labeled
analyte.
[0046] The sensing element is comprised of an electrode made by
electrochemically inert electroconductive material (such as
different carbon based materials, gold, different electroconductive
polymers) modified by immobilized binder (such as the analyte) that
is directly attached to the surface of the electrode and may be the
same as the analyte or as a detection compound.
BRIEF DESCRIPTION OF THE DRAWINGS
[0047] FIG. 1A illustrates an electron transfer at the surface of
an electrode. The electrode has an antigen (marked "Ag") affixed to
the electrode as its support and a labelled antibody/enzyme complex
(marked "Ab" and "Enzyme") is attached to the affixed antigen. Due
to the proximity of the enzyme to the electrode an electron is
transferred to the substrate and electrocatalytically the electrode
potential is changed by such electron transfer. Also shown in the
drawing, by discontinuous lines and a rough raised surface is a
semipermeable membrane across which an antigen may be diffused.
[0048] FIG. 1B illustrates a background electrode potential when
the antigen (marked "Ag") which is affixed to the electrode is not
linked to a labelled antibody/enzyme complex (marked "Ab" and
"Enzyme"). is attached to the affixed antigen. When the enzyme is
in near proximity to the electrode an electron is transferred to
the substrate and electrocatalytically the electrode potential is
changed by such electron transfer. However, when the labeled
antibody/enzyme complex becomes displaced (as shown in this
drawing) and the enzyme is distanced from the electrode, no
catalytic electron transfer occurs. Also shown in the drawing, by
discontinuous lines and a rough raised surface is a semipermeable
membrane across which an antigen may be diffused. A double arrow
shows one such opening through which an antigen (analyte) can
diffuse, but across which the antibody/enzyme complex cannot
move.
[0049] FIG. 2 illustrates an immunosensor apparatus according to
the invention comprising an electrochemical working electrode 1; an
electrochemical reference electrode 2; the diffusion membrane 3
which separates working electrode 1 from the external media; the
internal media 4 which may be a liquid or gel containing a
substrate for an enzyme-label as shown in FIG. 1A; an external
housing member 5 which fixes together the electrochemical working
electrode 1, the electrochemical electrode 2, the diffusion
membrane 3, the measuring device (the electrochemical interface) 6
which may be described as a digital voltmeter or an interface to
microprocessor which measuring device is connected to the sensing
element comprising the electrochemical working electrode 1 and the
supporting electrochemical reference electrode 2, and signal
recorder 7 which signal recorder may be described as an X-T
recorder, a microprocessor based data acquisition system with
digital display, or a personal computer.
[0050] FIG. 3 is a shematic illustrating continuous immunoassay
monitoring. In the presence of the analyte in the external media,
the analyte diffuses through the membrane and forms a complex with
an increase in potential. A decrease in the analyte concentration
in the external media results in dissociation of the complex and
leads to a decrease in the electrode potential.
DESCRIPTION OF SPECIFIC EMBODIMENTS
[0051] The present invention provides an alternative approach to
the development of immunosensors and is associated with
bio-electrocatalytic detection of the reversibly displacement of a
labeled detection compound that is reversibly bound to a binder
(which binder is an analyte or a compound which will bind to the
analyte) on an electrode surface, by free target analyte which will
either bind to the binder on the surface of the electrode or
compete with the binder for binding to the detection compound. In
this case, the electrode is separated from the external media by
porous membrane permeable for analyte and impermeable to higher
molecular weight compounds such as the enzyme-labeled detection
compound. Therefore, the labeled detection compound does not
diffuse to the external media. A decrease in the concentration of
target analyte in external media results in a new (target
analyte)/(labeled detection compound) ratio and a new equilibrium
with respect to how much of the enzyme-labeled detection compound
is bound to the binder on the electrode. Such a decrease leads to
release of analyte that is bound to either the binder or to the
detection compound and to the re-binding of some labeled detection
compound to the binder on the surface of the electrode. Therefore,
continuous monitoring of the concentration of the analyte in a
sample source is allowed (FIG. 1).
[0052] As shown in FIG. 2, the working electrode 1 serves for
potentiometric measurement. It consists of an electro-conductive
material modified by the immobilized analyte. The reference
electrochemical electrode 2 can be fixed inside the inert body 5 or
be immersed into it together with sensing element. It can be
represented by high impedance voltmeter or by interface which
converts voltage signal to the format suitable for acquisition by
microprocessor or personal computer. The signal recorder 7 serves
to measure and visualize the initial rate of the increase of the
electrode potential of the sensing element. It can be represented
by simply X-T recorder, or by microprocessor based digital data
acquisition system, or by personal computer supported with special
software.
[0053] The advantage of the sensing device having a semipermeable
membrane lies in its potential to provide continuous on line
monitoring of analyte. The ability of the sensing device to detect
analyte over a wide concentration range allows its use for on-line
measurements of concentration for different analytes. FIG. 1
presents the overall scheme of the electrode function.
[0054] The working electrode is represented by an
electro-conductive highly dispersed material such as flat or
dispersed carbon, graphite, carbon black, conductive dispersed
pyrolytic products, conductive metal oxides, metal and metal
powders, semiconductor materials, dispersed conductive polymers
with binders (analytes or corresponding antibodies, antigens, or
haptens for such analytes) that are immobilized on the surface of
the electrode material.
[0055] The liquids used in the invention are water, buffer
solutions, and aqueous sample diluents. Preferred liquids are
buffer solutions such as phosphate buffered saline, borate buffered
saline, acetate buffered saline, TRIS saline and the same buffer
solutions containing detergents such as Tween, Triton etc. in
different concentrations.
[0056] The enzyme-labels used in the invention are
electrocatalytically active oxidoreductases represented by but not
limited to laccase (substrate oxygen); lactate dehydrogenase
(substrate lactate); horseradish peroxidase, cytochrome c
peroxidase, fungal peroxidases, lactoperoxidase, microperoxidase,
chloroperoxidase (substrate hydrogen peroxide); hydrogenase
(substrates hydrogen, proton); D-fructose dehydrogenase (substrate
fructose), methylamine dehydrogenase (substrate methylamine);
flavocytochrome c552 (substrate sulfide); succinate dehydrogenase
(substrates succinate, fumarate); fumarate reductase (substrate
fumarate); alcohol dehydrogenase (substrate ethanol); D-gluconate
dehydrogenase (substrate gluconate); cellobiose dehydrogenase
(substrate cellobiose); and ascorbate oxidase (substrate
oxygen).
[0057] In a preferred aspect, the invention relates to
immunoelectrochemical analysis employing laccase as the enzyme
label. Laccase possesses strong electrocatalytic properties in that
it can catalyze the electroreduction of oxygen (Berezin et al.,
Doklady Phys. Chem., 240:455 (1978, translated from Russian):
O.sub.2+4H.sup.++4e.sup.-.fwdarw.2H.sub.2O
[0058] When laccase is utilized as the enzyme label the donor
substrate is an electron moving directly from the an electrode
which is in the proximity of the laccase enzyme and the second
substrate is molecular oxygen.
[0059] The ability of the enzyme laccase to catalyze
electroreduction of oxygen via a direct mechanism allows the
detection of the biospecific interaction of a laccase-labeled
receptor, or antibody, with a ligand-modified electrode. The
potential established on the electrode coated with immobilized
laccase is close to the equilibrium oxygen potential, and the shift
in potential occurring in the presence of the immobilized enzyme
can be as high as 400 mV. The bioaffinity interaction occurring on
the electrode surface can therefore be determined by using a
laccase-labeled bioconjugate. Formation of a complex between the
laccase-labeled antibody and the antigen on the electrode surface
results in a considerable (>300 mV) change in the electrode
potential.
[0060] The change in electrode potential is due to the transfer of
electrons directly form the electrode as a reaction substrate for
laccase which react with the other substrate for laccase which is
atmospheric oxygen, in that electrons are transferred directly from
the electrode to the active site of the enzyme label. A composite
carbon material containing a polyethyleneimine-based polymer can be
used to eliminate nonspecific interactions between the reaction
components and the electrode surface.
[0061] The potentiometric detection of an analyte does not depend
on the electrode surface area. The electrode response is a function
of the fraction of electrode surface that is covered by the
electro-active label (laccase). This fact opens a possibility for
miniaturization immuno-electrodes without effecting of their
sensitivity. One of the important advantages of the
bio-electrocatalytic detection approach in immunoassay is
associated with the fact that the reaction does not involve low
molecular weight substrates (except dissolved oxygen).
[0062] Several general practical advantages of the potentiometric
immuno-sensors according to the invention can be summarized in five
engineering issues: (i) potentiometric detection has a potential
for a great degree of miniaturization of sensing elements; (ii) the
manufacturing procedure of the sensing elements can easily be
adapted for mass production being compatible with techniques such
as screen-printing or ink-jet dispensing; (iii) it is possible to
design low cost and thus, disposable sensing elements; (iv) the
measuring equipment is simple consisting of a high impedance
voltmeter.
[0063] In a preferred aspect, the invention provides a
potentiometric immunosensor based on mediatorless
bioelectrocatalysis that utilizes the laccase enzyme as an
electrocatalyst-label. The electrocatalytic property of the enzyme
in the reaction of oxygen electroreduction (reaction 1) allows the
detection of the bio-specific interaction of a laccase-labeled
receptor, or antibody, with a ligand modified electrode. Analysis
was performed in a competitive scheme, and a single measurement was
made within 20 minutes. Such a potentiometric immunoassay does not
require an electrochemically active mediator. The reaction
substrates are atmospheric oxygen and electrons that are
transferred directly from the electrode to the oxygen molecule via
the active site of the enzyme. Insulin may be used as a model
analyte as illustrated by the examples below. Furthermore, a higher
rate of electrode potential shift can be achieved by employing a
high concentration of immuno-conjugate. A competition for binding
with immobilized immuno-species for high concentration of
immuno-conjugate can require a relatively high concentration of the
analyte.
[0064] In one aspect of the invention the laccase enzyme label is
bound to a binder (antibody, antigen or hapten, that is referred to
above as a detection compound) for an analyte which detection
compound is also bound by a binder on the electrode. The analyte
will cause the enzyme label/detection compound to disassociate from
the binder on the electrode and bind to the analyte to cause a
change in potential of the electrode. Depending upon dynamic
changes in the concentration of analyte that moves across the
semipermeable membrane of the sensor which contains the enzyme
label/ligand and electrode, differing measurements of electrode
potential are observed. For example, at higher concentrations of
analyte more of the enzyme label/detection compound is
disassociated from the electrode to bind to the analyte and at
lower concentrations of the analyte more of the enzyme
label/detection compound binds to the binder on the electrode.
Since measurements of the increase or decrease in electrode
potential can be made simultaneously with the biospecific
interaction of the affinity components on the electrode surface,
continuous measurements can be performed in the kinetic mode. This
also allows for a significant reduction in analysis time since the
rate of change in potential can be utilized to determine the
changes in concentration as well as equilibrium measurements of
potential.
[0065] In general, the amplitude of signals produced by working
electrodes according to the invention is determined by the extent
to which the electrode surface is coated with laccase-conjugated
molecules, and not by the total amount of conjugated laccase bound
to the electrode. A carbon composite can be used as an electrode
material which possess a considerably large effective surface area,
but higher sensitivity can be attained by the use of an electrode
materials with a lower, or low, effective surface. The pH of the
assay solution must be high enough to promote antigen-antibody
complex formation. At the same time, it should not exceed the
optimal value for the enzymatic activity, which is the case of
laccase activity is about pH 5.5, but may exceed the optimal value
for laccase activity somewhat. For example, an increase in pH above
5.5 when using laccase can result in a sharp decrease in the rate
of potential change in the presence of the laccase-antibody
conjugate, but may be as high as 6.5 where laccase from Coriolus
sp. still retains 30-50% of maximal activity. Since laccase from
different sources may differ in pH for their optimium activity, a
laccase may be selected from a particular source when a particular
optimal pH is desired. For example, laccase from certain fungi has
a pH optimal activity near 7.0. Electrodes according to the
invention, as described above do not have changes in the increase
of the electrode potential over the temperature range of 30.degree.
C.-37.degree. C., and dry IgG-modified electrodes will generally
retain their activity during storage for at least 2 months at
4.degree. C.
[0066] In another aspect the invention provides a method and
apparatus for reagentless immunoassay is described, in that no
reagent beyond the self-contained sensor apparatus is required and
the self-contained sensor apparatus may be used for multiple
measurements.
[0067] In a preferred embodiment of the invention the apparatus
comprises an immunosensor have the general immunosensor design
shown in FIG. 2, which includes the working electrode 1; the
reference electrochemical electrode 2; a diffusion membrane which
separates the working electrode 1 from the external media; the
internal media 4 which is liquid or gel containing a substrate for
an enzyme-label; an external housing member 5 which fixes together
the working electrode 1, the electrochemical reference electrode 2,
the diffusion membrane 3, the measuring device (the electrochemical
interface) 6 which is a digital voltmeter or an interface to
microprocessor which is connected to the sensing element and the
supporting electrochemical probe 2, and signal recorder 7 which is
an X-T recorder, microprocessor based data acquisition system with
digital display, or personal computer. The working electrode 1
serves for potentiometric measurement. It consists of an
electro-conductive material modified by the immobilized analyte.
The supporting electrochemical reference electrode 2 can be fixed
inside the external housing member 5 or be immersed into it
together with the sensing element. It can be represented by high
impedance voltmeter or by interface which converts voltage signal
to the format suitable for acquisition by microprocessor or
personal computer. The signal recorder 7 serves to measure and
visualize the initial rate of the increase of the electrode
potential of the sensing element. It can be represented by simply
X-T recorder, or by microprocessor based digital data acquisition
system, or by personal computer that is set to record and analyze
the measurement results.
[0068] The above described apparatus may have multiple electrodes
where the different electrodes are for different analytes. Thus,
multiple analytes in a single sample may be analyzed for by
utilizing the multiple electrodes.
[0069] Further, a Sandwich Assay Scheme can be used for continuous
immunoassy monitoring. In the case where both primary and secondary
(labeled) antibody are low affinity antibodies, the formation of a
complex Ab1-Analtye-Ab2-Laccase may be reversable.
[0070] Having described the invention, the following non-limiting
examples are given to illustrate specific techniques and
applications of the principles of the invention. which can be used
to carry out the invention. These specific examples are for
illustrative purposes only and are not intended to limit the scope
of the invention described in this application.
EXAMPLE 1
Preparation of a Laccase Enzyme Conjugate
[0071] Laccase from Coriolus sp. was obtained as described by
Ghindilis et al., Biochemistry (translated from Russian:
Biokhimiya) 53:635-639 (1988). Pig insulin was obtained as a
commercial product (for example, Sigma, St. Louis, Mo., U.S.A.).
The laccase-insulin conjugate was synthesized by using the general
procedure of Nakane et al., J. Hist and Cyto. Chem., 22 1084-1091
(1974) for the preparation of peroxidase conjugates. This procedure
was modified as follows: a solution of NaIO was added to the
solution of laccase in distilled water (1 mg ml.sup.-1) to give a
final concentration of 1.0 M. The mixture was incubated for 30
minutes, in the dark, at 18-22.degree. C., and dialyzed against
0.1M sodium acetate buffer, pH 4.5 at 4.degree. C. for 14-16 hours.
Insulin was gradually added to the enzyme solution to give a molar
ratio of 3:2. The mixture was incubated for 3 hours at
18-22.degree. C. The pH of the reaction medium was maintained
between 8.8 and 9.0. The conjugate obtained was dialyzed against
0.1 M phosphate buffer, pH 6.5, at 4.degree. C., for 16-18 hours,
and stored in 50% glycerol at -18.degree. C. EXAMPLE 2
Preparation of a Working Electrode
[0072] Graphite Ink, EXP 741801, obtained by Ercon (Wareham,
Mass.), was deposited on a plastic strip (2.times.30 mm) and then
dried at room temperature for 14-16 h. The electrode body was then
encapsulated with fast dry enamel 720, obtained from Maybeline Inc.
(New York, N.Y.). The tips of the electrode (2.times.2 mm) remained
non-encapsulated to serve as a working electrode surface and as a
connector to the electrical circuit. The electrode was then
pretreated, by forced polarization, in a three electrode
electrochemical cell under an electrode potential of -0.8 V versus
an Ag/AgCI reference electrode for 20 min. A solution of monoclonal
anti-insulin antibodies (20 .mu.g/ml) in phosphate buffered saline
(PBS) was then placed onto the working tip of the electrode and
dried to achieve immobilization by physical absorption. Monoclonal
antibodies were developed by Biocon Inc. (Rockville, Md.), from the
ATCC HB127 hybridoma line obtained from ATTC (Rockville, Md.). The
electrode was then incubated in a solution of trypsin inhibitor
(0.1 mg/ml) in PBS to block free sites of non-specific binding for
4h at room temperature. The electrode was then pre-incubated in the
solution of laccase-insulin conjugate (0.1 mg/ml) in 0.1 M
phosphate buffer, pH 6.2, containing 1 mg/ml human serum albumin
(HSA) at 37 C., for 16 h, to achieve an attachment of the
laccase-insulin conjugate to the electrode surface.
EXAMPLE 3
Analysis of Analyte Insulin Solution With a Working Electrode
[0073] The electrode was then placed into the sensing apparatus, as
described in FIG. 2. The internal medium of the housing member
contained a solution of laccase-insulin conjugate (5 .mu.g/ml) in
0.1 M phosphate buffer, pH 6.2, containing 1 mg/ml HSA. The sensing
apparatus was then placed in a contact with an external media (0.1
M phosphate buffer, pH 6.2, containing 1 mg/ml HAS) containing
insulin analyte.
[0074] Atmospheric oxygen, which is a substrate for laccase,
diffuses to the internal media through the external media. The
potential of the working electrode, in the absence of insulin in
the external media (antibody/laccase complex is bound to insulin
that is affixed to the electrode) was close to O.sub.2/H.sub.2O
potential and was about 350 mV (vs Ag/AgCl electrode). The addition
of insulin into the external media resulted in a shift of the
potential towards the background carbon electrode potential (100
mV). The potential change was proportional to the concentration of
insulin in the external media in a wide range of insulin
concentrations.
[0075] As a reference, an Ag/AgCl electrode was used. Potential
changes were measured by means of a high impedance voltmeter.
* * * * *