U.S. patent application number 15/944148 was filed with the patent office on 2018-08-16 for electrochemical analyte detection apparatus and method.
This patent application is currently assigned to AgaMatrix, Inc.. The applicant listed for this patent is AgaMatrix, Inc.. Invention is credited to Ian Harding, Sridhar Iyengar, Richard Williams.
Application Number | 20180231552 15/944148 |
Document ID | / |
Family ID | 40281828 |
Filed Date | 2018-08-16 |
United States Patent
Application |
20180231552 |
Kind Code |
A1 |
Harding; Ian ; et
al. |
August 16, 2018 |
Electrochemical Analyte Detection Apparatus and Method
Abstract
A method and apparatus for electrochemical detection of analyte
in a sample makes use of a binding interaction and relies on the
discovery that asymmetric distribution of a redox enzyme between
two electrodes that occurs when a redox enzyme-containing reagent
is immobilized at the surface of one electrode can be detected as a
chemical potential gradient arising from an asymmetry in the
distribution of oxidized or reduced redox substrate. This chemical
potential gradient can be detected potentiometrically by observing
the potential difference between the electrodes in an open circuit,
or amperometrically by observing the current flow between the
electrodes when the circuit is closed. In both cases, the
observation of asymmetry can be done without the application of an
external potential or current to the electrodes.
Inventors: |
Harding; Ian; (Wells,
GB) ; Iyengar; Sridhar; (Salem, NH) ;
Williams; Richard; (Somerville, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AgaMatrix, Inc. |
Salem |
NH |
US |
|
|
Assignee: |
AgaMatrix, Inc.
Salem
NH
|
Family ID: |
40281828 |
Appl. No.: |
15/944148 |
Filed: |
April 3, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15017787 |
Feb 8, 2016 |
9939440 |
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15944148 |
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12179446 |
Jul 24, 2008 |
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15017787 |
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60952099 |
Jul 26, 2007 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/001 20130101;
G01N 2333/90 20130101; G01N 33/5438 20130101; G01N 27/4166
20130101; G01N 2333/902 20130101; G01N 33/573 20130101 |
International
Class: |
G01N 33/573 20060101
G01N033/573; C12Q 1/00 20060101 C12Q001/00; G01N 33/543 20060101
G01N033/543; G01N 27/416 20060101 G01N027/416 |
Claims
1. A method for determining an analyte in a sample, comprising the
steps of: (a) introducing the sample to a test cell comprising
first and second electrodes, a mobile test reagent comprising a
redox enzyme portion, and reagents that interact with the mobile
test reagent and/or the analyte, such that the mobile test reagent
has a first distribution relative to the electrodes when analyte is
present in the sample and a second distribution relative to the
electrodes when analyte is not present sample, one of said first
and second distributions being asymmetric with respect to a line
between the electrodes, and the other of the first and second
distributions being symmetric or less asymmetric with respect to
the line between the electrodes, (b) supplying a redox substrate
for the redox enzyme in the test cell, said redox substrate being
acted upon by the redox enzyme to produce a chemical potential
gradient between the first and second electrodes, wherein the
magnitude of the chemical potential gradient is determined by the
distribution of the mobile test reagent and thus on the presence of
analyte, and (c) detecting the chemical potential gradient between
the first and second electrodes to determine analyte in the sample,
wherein the formation and detection of the chemical potential
gradient are performed without application of an external potential
or current.
2. The method of claim 1, wherein the test cell comprises as one of
the reagents that interact with the analyte an immobilized test
reagent disposed on the surface of the first electrode, said
immobilized test reagent comprising an analyte binding portion, and
the mobile test reagent comprises an analyte-binding portion and
the redox enzyme portion, whereby if analyte is present in the
sample in the test cell, at least a portion of the mobile test
reagent becomes immobilized on the first electrode thereby creating
an asymmetric distribution of redox enzyme concentration between
the first and second electrodes when analyte is present.
3. The method of claim 2, wherein the analyte binding portion of
the immobilized test reagent and the analyte are an
antibody-antigen pair or antibody/hapten pair.
4. The method of claim 3, wherein the redox enzyme and the redox
substrate comprises glucose and a mediator compound.
5. The method of claim 2, wherein the analyte binding portion of
the immobilized test reagent is a drug receptor.
6. The method of claim 5, wherein the redox enzyme and the redox
substrate comprises glucose and a mediator compound.
7. The method of claim 2, wherein the analyte binding portion of
the immobilized test reagent is a hormone receptor.
8. The method of claim 7, wherein the redox enzyme and the redox
substrate comprises glucose and a mediator compound.
9. The method of claim 1, wherein the analyte binding portion of
the immobilized test reagent and the analyte are an
antibody-antigen pair or antibody/hapten pair.
10. The method of claim 9, wherein the redox enzyme and the redox
substrate comprises glucose and a mediator compound.
11. The method of claim 1, wherein the analyte binding portion of
the immobilized test reagent is a drug receptor.
12. The method of claim 11, wherein the redox enzyme and the redox
substrate comprises glucose and a mediator compound.
13. The method of claim 1, wherein the analyte binding portion of
the immobilized test reagent is a hormone receptor.
14. The method of claim 13, wherein the redox enzyme and the redox
substrate comprises glucose and a mediator compound.
15. An apparatus for determining an analyte in a sample comprising
a test strip comprising first and second electrodes disposed to
contact a test cell for receiving sample, said test strip further a
mobile test reagent comprising a redox enzyme portion, and reagents
that interact with the mobile test reagent and/or the analyte, such
that the mobile test reagent has a first distribution relative to
the electrodes when analyte is present in the sample and a second
distribution relative to the electrodes when analyte is not present
sample, one of said first and second distributions being asymmetric
with respect to a line between the electrodes, and the other of the
first and second distributions being symmetric or less asymmetric
with respect to the line between the electrodes, and a circuit for
detecting the symmetric or asymmetric distribution of the mobile
test reagent in the test cell in the absence of an applied external
potential or current, and means for communicating the detected
distribution to a user.
16. The apparatus of claim 15, wherein the circuit detects the
potential difference between the first and second electrode.
17. The apparatus of claim 15, wherein the circuit detects current
flow in a circuit including the electrodes.
18. The apparatus of claim 15, wherein the first and second
electrodes are in a facing configuration.
19. The apparatus of claim 15, wherein the first and second
electrodes are in a side-by-side configuration.
20. The apparatus in accordance with claim 15, wherein, the test
cell comprises as one of the reagents that interact with the
analyte an immobilized test reagent disposed on the surface of the
first electrode, said immobilized test reagent comprising an
analyte binding portion, and the mobile test reagent comprises an
analyte-binding portion and the redox enzyme portion, whereby if
analyte is present in the sample in the test cell, at least a
portion of the mobile test reagent becomes immobilized on the first
electrode thereby creating an asymmetric distribution of redox
enzyme concentration between the first and second electrodes when
analyte is present.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/952,099 filed Jul. 26, 2007, which application
is incorporated herein by reference for all purposes.
BACKGROUND OF THE INVENTION
[0002] This application relates to an apparatus and method for
electrochemical detection of an analyte in a sample. The invention
utilizes a specific binding relationship between the analyte and at
least one reagent provided in the apparatus as a means for
detecting the analyte.
[0003] Analysis methods in which specific binding between an
analyte and a reagent forms the basis for the assay are known. For
example, in enzyme-linked immunoassay (EIA or ELISA) procedures, a
sandwich is formed between an immobilized antibody and a mobile
enzyme-antibody reagent when analyte is present through the
interaction of the analyte with the antibody components. This
results in the immobilization of the enzyme. The subsequent
detection of immobilized enzyme is therefore indicative the
presence of analyte in a test solution. (See U.S. Pat. Nos.
3,654,090, 4,169,012, and 4,642,285) Similar sandwich assays are
used in chromatographic immunoassays where a colored tag (for
example a colored latex bead) on the mobile reagent becomes trapped
on a substrate at a defined location to produce a pattern that
indicates the presence of analyte in a sample. (See U.S. Pat. Nos.
4,943,522, 5,656,503, 5,712,172 and 5,766,961) Analysis methods
that depend on specific binding between an analyte and a reagent
may also take the form of competition assays, in which the
formation of a complex involving a labeled reagent is inhibited in
the presence of analyte in an analyte-concentration dependent
manner (See U.S. Pat. Nos. 4,868,131, 5,981,298 and 5,989,921), or
displacement assays in which a pre-existing complex involving a
labeled reagent is disrupted in the presence of analyte in an
analyte-concentration dependent manner. (See U.S. Pat. Nos.
4,746,631 and 6,020,209).
[0004] Immunoassays in which detection of the analyte depends on an
electrochemical measurement are also known. U.S. Pat. No. 5,149,630
discloses an assay in which the extent to which the transfer of
electrons between an enzyme substrate and an electrode, associated
with the substrate reaction, is perturbed by complex formation or
by displacement of any ligand complex relative to unbound
enzyme-labelled component. This determination is made in the
presence of an applied potential. An applied potential is also used
to measure current in the assay device disclosed in U.S. Pat. Nos.
5,198,367, 5,427,912. U.S. Pat. No. 5,494,831 discloses the
application of a current and the measurement of changes in
impedance that result in binding.
SUMMARY OF THE INVENTION
[0005] The present invention provides a new method and apparatus
for electrochemical detection of analyte in a sample that makes use
of a binding interaction. The invention relies on the discovery
that asymmetric distribution of a redox enzyme between two
electrodes that occurs when a redox enzyme-containing reagent is
immobilized at the surface of one electrode can be detected as a
chemical potential gradient arising from an asymmetry in the
distribution of oxidized or reduced redox substrate. This chemical
potential gradient can be detected potentiometrically by observing
the potential difference between the electrodes in an open circuit,
or amperometrically by observing the current flow between the
electrodes when the circuit is closed. In both cases, the
observation of asymmetry can be done without the application of an
external potential or current to the electrodes.
[0006] In a first embodiment of the invention, a sandwich type
assay is utilized. In this embodiment, a sample to be tested for
analyte is introduced to a test cell in the presence of a mobile
test reagent. The test cell comprises a first and a second
electrode. The first electrode has immobilized on the surface
thereof an immobilized test reagent. The mobile test reagent
comprises an analyte-binding portion and a redox enzyme portion,
and the immobilized test reagent comprises an analyte binding
portion. If analyte is present in the sample in the test cell, at
least a portion of the mobile test reagent becomes immobilized on
the first electrode thereby creating an asymmetry in redox enzyme
concentration between the first and second electrodes when analyte
is present. A redox substrate for the redox enzyme in the test cell
is also supplied.
[0007] The redox substrate is acted upon (oxidized or reduced) by
the redox enzyme. If there is an asymmetry in redox enzyme
concentration between the first and second electrodes (i.e. when
analyte is present in the sample), this results in a chemical
potential gradient between the first and second electrodes. This
chemical potential gradient is detected to determine analyte in the
sample. In this embodiment, the greater the asymmetry and the
resulting potential gradient, the greater the amount of analyte in
the sample.
[0008] In a second embodiment of the invention, a competition or
displacement type of assay is utilized. In this assay, the redox
enzyme is coupled to analyte or an analog of the analyte that can
bind to a common immobilized test reagent. Optionally, the redox
enzyme is provided already bound to the electrode via the
immobilized test reagent. When sample is added, analyte present in
the sample will compete with the redox enzyme for the binding sites
provided by the immobilized test reagent resulting in a reduction
in the amount of asymmetry that would occur is all of the binding
sites were occupied by redox enzyme.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIGS. 1A and B show schematic representations of reduced
mediator distribution between two electrodes when redox enzyme
distribution is asymmetric (FIG. 1 A) or symmetric (FIG. 1B).
[0010] FIGS. 2A and B illustrate the determination of asymmetric
enzyme distribution at two electrodes using amperometry.
[0011] FIG. 3 shows a sandwich assay format useful in the present
invention.
[0012] FIGS. 4 and 5 shows competition/displacement assay format
useful in the present invention.
[0013] FIGS. 6A-C shows construction of a test cell with facing
electrodes.
[0014] FIGS. 7A-C show examples of side-by-side electrode
configurations.
[0015] FIG. 8 shows an external view of a meter in accordance with
the invention.
[0016] FIG. 9 shows a circuit usable in the meter of the
invention.
[0017] FIG. 10 shows potential difference in open circuit as a
function of time for different levels of asymmetry in enzyme
distribution between two electrodes.
[0018] FIG. 11 shows current in short circuit as a function of time
for different levels of asymmetry in enzyme distribution between
two electrodes.
[0019] FIG. 12 shows the correlation of peak potential difference
with ratio of electrode enzyme activities
[0020] FIG. 13 shows the correlation of peak current with ratio of
electrode enzyme activities
[0021] FIG. 14 shows a schematic plot of a known total enzyme
E.sub.t on x-axis and measured I.sub.300 on the y-axis.
[0022] FIG. 15 shows a schematic plot of a known E.sub.t on x-axis
and measured I.sub.0 on the y-axis.
[0023] FIG. 16 shows a plot of how log (R) affects the slope of
I.sub.0 versus E.sub.t which is used to derive parameters a and
b.
[0024] FIG. 17 shows a plot of log (R) affects the intercept of
I.sub.0 versus E.sub.t which is used to derive parameters c and
d.
[0025] FIG. 18 shows the correlation between the actual amount of
an enzyme and the estimated amount of enzyme as determined in
accordance with the invention.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0026] As used in the specification and claims of this application,
the following definitions should be applied:
[0027] (a) "analyte" refers to a material of interest that may be
present in a sample. Analytes that are detectable in the present
invention are those that can be associated in a specific-binding
interaction with at least one other reagent to that they can
participate in a sandwich, competition or displacement assay
configuration as described herein. Examples of analytes include
antigens or haptens such as peptides (for example hormones),
proteins (for example, enzymes), drugs, pesticides, microorganisms,
antibodies, and nucleic acids that can participate in sequence
specific hybridization reactions with a complementary sequence.
[0028] (b) "analyte-specific enzyme component" or reagent refers to
a reagent that includes both an analyte-binding portion and a redox
enzyme portion. An analyte-specific enzyme component is suitably
used as a mobile reagent.
[0029] (c) "determination of an analyte" refers to qualitative,
semi-quantitative and quantitative processes for evaluating a
sample. In a qualitative evaluation, a result indicates whether or
not analyte was detected in the sample. In a semi-quantitative
evaluation, the result indicates whether or not analyte is present
above some pre-defined threshold. In a quantitative evaluation, the
result is a numerical indication of the amount of analyte
present.
[0030] (d) the term "redox enzyme" refers to an enzyme that
oxidizes or reduces a substrate. Such enzymes may generally be
known as oxidases, peroxidases, reductases, or dehydrogenases.
Enzymes such as glucose oxidase, and various peroxidases are
commonly used in the analytical devices, and therefore the
preparation of these enzymes in stable form is well known.
[0031] (e) the term "redox substrate" refers to a compound or
combination of compounds that interact with the redox enzyme to
produce a chemical potential gradient. In some cases, the enzyme
substrate may directly produce a redox active species sufficient to
create the chemical potential gradient. In others, a secondary
compound may be needed. For example, in the case of glucose
oxidase, the interaction with glucose to produce gluconolactone and
reduced enzyme produces the chemical potential gradient when the
reduced enzyme is oxidized by a mediator compound, which is the
actual redox active species in the chemical potential gradient.
Thus, in this case the "redox substrate" is the combination of the
substrate compound glucose and the mediator compound.
[0032] (f) the term "chemical potential gradient" refers to a
concentration gradient of a redox active species. It will be
appreciated that more rigorously, the potential gradient arises
from a gradient in the ratio of reduced to oxidized species between
the electrodes, however, the idea of a concentration gradient of
one species is more easily visualized and is therefore used here.
When such a gradient is present between two electrodes, a potential
difference is detectable if the circuit is opened, and a current
will flow until the gradient dissipates when the circuit is closed.
It will be appreciated that the chemical potential gradient is
transient in the devices of the invention, and that the
distribution of the redox active species will even out over time,
when new redox active species stops being created. The term
"chemical potential gradient" as used herein refers only to this
transient gradient that arises from the asymmetry of the
distribution of redox enzyme and not to any potential gradient that
arises from the application of a potential difference or current
flow between the electrodes.
[0033] (g) the phrase "detecting the chemical potential gradient
between the first and second electrodes" refers to the detection of
the chemical potential gradient in either an open or a closed
circuit, using either potentiometric or amperometric
measurements.
[0034] (h) the terms "enzyme activity" and "enzyme concentration"
are used as equivalent terms herein, although it will be
appreciated that in ordinary usage they may have different
meanings. Activity of an enzyme provides a quantitative measure of
the catalytic capability of an enzyme. This depends not only on the
physical amount of the enzyme present in the volume (i.e. the
concentration), but also on the conditions which affect the
catalytic efficiency of the enzyme. The present invention actually
measures asymmetry in enzyme activity, since the presence of
inactive enzyme will not produce an asymmetry in redox substrate.
However, since it is desirable to control the quality of the enzyme
and the conditions, this is in effect also a measurement of
asymmetry in enzyme concentration.
[0035] (i) the term "immobilized on the first electrode" refers to
immobilization directly or indirectly on the surface of the
electrode, provided that the material immobilized becomes
immobilized in volume associated with the first electrode and
closer to the first electrode than the second electrode. For
example, in the case of the formation of an
electrode-antibody-analyte-antibody-enzyme sandwich, the enzyme is
considered to be "immobilized on" the electrode even though there
are several intervening moieties in the sandwich.
[0036] (j) the term "immobilized test reagent" refers to the
component of a sandwich or competition/displacement reaction that
is associated with an electrode when performing the assay of the
invention. Immobilization may be through the formation of a
chemical bond (covalent or non-covalent) between the immobilized
test reagent and the surface of the electrode, or it may be a
physical association as through the placement of the immobilized
test reagent within a gel or membrane disposed on the surface of
the electrode. The immobilized test reagent comprises a binding
moiety which interacts with analyte to produce a change in the
distribution of the redox enzyme (as part of the analyte-binding
enzyme component) when analyte is present in a sample. In some
embodiments, the immobilized reagent will be immobilized during
initial manufacture of the test apparatus. In other embodiments,
immobilization will occur in situ after addition of the sample.
Thus, the term immobilized test reagent refers to any circumstance
in which the structure: [0037] electrode-(link).sub.n-reagent with
binding site for analyte is obtained, where n is 0 or an integer of
1 or greater. The latter option allows production of one set of
generic devices in which the linking agent is not analyte specific,
with the addition of reagent that binds to the link and the analyte
for any given analyte-specific test. This additional reagent could
be added to the test strip at manufacture, or to the sample prior
to application to the test device.
II. Theory of the Invention
[0038] For convenience, the theoretical basis for the invention
will be discussed in the context of glucose oxidase as the redox
enzyme and a combination of glucose and a mediator as the redox
substrate. Nothing in this discussion should be taken as an
indication, however, that the invention is limited to use with
these materials.
[0039] In common glucose measurement systems, enzyme present in the
sample cell oxidizes glucose to gluconolactone, and the enzyme is
reduced. Oxidized mediator (for example ferricyanide) reacts with
the reduced enzyme to regenerate the oxidized form of the enzyme,
and produce reduced mediator. This process continues until either
glucose or oxidized mediator is exhausted. If the enzyme is
distributed asymmetrically within the sample cell, then the
production of reduced mediator is also asymmetric, and the
resulting asymmetry in the distribution of reduced mediator
persists for a period of time (determined by diffusion parameters)
even after exhaustion of the limiting reagent.
[0040] When reduced mediator is present in a solution between two
electrodes, the potential difference between the two electrodes is
given by the Nernst equation
E = RT F X log ( [ med red ] electrode 1 [ med ox ] electrode 1 X [
med ox ] electrode 2 [ med red ] electrode 2 ) ##EQU00001##
where E is the potential difference between electrodes 1 and 2, R
is the gas constant, T is the absolute temperature and F is the
Faraday constant. When there is no difference in the concentration
of reduced mediator at the two electrodes (i.e. no chemical
potential gradient) because there is no asymmetry, the mediator
terms reduce to 1, log 1=0, and so the potential difference is 0.
As the asymmetry increases, the potential difference increases.
Furthermore, if the system is designed such that the oxidized
mediator concentration at the time of measurement is large compared
to the amount of reduced mediator (either due to a large excess of
oxidized mediator initially or rapid taking of the measurement or
both) then the oxidized mediator concentration at the two
electrodes is essentially equal, and the equation can be simplified
to:
E = RT F X log ( [ med red ] electrode 1 [ med red ] electrode 2 )
##EQU00002##
[0041] FIGS. 1A and B show schematic representations of reduced
mediator distribution between two electrodes (1, 2) when redox
enzyme distribution is asymmetric (FIG. 1 A) or symmetric (FIG.
1B). The equations above can be used to quantitate the ratio of
concentrations of enzyme at two electrodes 1 and 2 using
potentiometry. The two electrodes are connected in open circuit,
and the potential difference between them is measured. If enzyme 3
is more active or concentrated at one electrode than the other
(i.e., it has an asymmetric distribution as shown in FIG. 1A), the
concentration of reduced mediator will be higher at that electrode
than the other, giving rise to a electrical potential difference
between the electrodes. If equal activity or concentrations of
enzyme are present at both electrodes (i.e., it has a symmetric
distribution as shown in FIG. 1B), the concentration of reduced
mediator at each electrode will be equal, and the electrodes will
be at equal electrical potential and the measured potential
difference will be 0.
[0042] If, instead of potentiometry, the electrodes are connected
in short-circuit, the asymmetric distribution of enzyme activity
will result in an asymmetric distribution of reduced mediator, that
can be observed by following current when the potential difference
between the electrodes is forced to zero by closing the circuit
between them. If reduced mediator is generated by enzyme activity
and diffuses to an electrode, current will flow and sufficient
mediator will be reoxidized, such that the reduced mediator
concentration is equal at both electrodes. The current flow will be
proportional to the difference in flux of reduced mediator to the
two electrodes.
[0043] FIGS. 2A and B illustrate the use of these principles to
quantify the ratio of concentrations of enzyme at two electrodes,
using amperometry. The two electrodes are connected in short
circuit, and the current flowing between them is measured. As
electrical potential difference between the electrodes is
constrained to zero, mediator will be reduced or oxidized at the
electrodes when necessary to maintain an equal chemical potential
at each electrode. As reduced mediator is oxidized at one
electrode, current will flow and an equivalent quantity of oxidized
mediator will be reduced at the other electrode. If the enzyme
activity at each electrode is equal, no electron transfer will be
necessary to keep the chemical potentials balanced, so no current
will flow. If more enzyme activity is present at one electrode than
the other, reduced mediator will be consumed at that electrode and
produced at the other electrode to maintain a balanced chemical
potential, and current will flow.
III. Practical Applications
[0044] The theory of the present invention is applicable to the
practical application of determining analytes in a sample. This can
be done in various binding formats. In general, two
analyte-specific chemical components are utilized, and these are
selected in to correspond to the analyte to be determined. In
general, it is desired to have at least one of the components
(generally the analyte-receptor) be highly specific for the analyte
while the other component can be less specific in its binding.
Various non-limiting combinations are set forth in Table 1.
TABLE-US-00001 binding component of Analyte- Analyte Analyte
receptor specific enzyme disease-specific disease associated-
antibody that recognizes human antibody, for example an antigen,
such as peptide antibodies, aptamers antibody to influenza, epitope
found on an disease causing organism influenza of interest,
aptamers amplified nucleic acid nucleic acid nucleic acid sequence
complementary sequence including a complementary to the to the
known primer suspected target and a suspected target known primer
hormone hormone receptor, hormone receptor, apatamer or apatamer or
antibody antibody drug drug receptor, aptamer or hormone receptor,
apatamer or antibody antibody pesticides pesticide binding
pesticide binding proteins, apatmers proteins, aptamers, antibodies
that recognize the antibodies to pesticides antibodies to
pesticides
[0045] As will be understood in the art, the "antibody"
incorporated in the device can be a complete antibody such as an
immunoglobulin, or it may be a engineered binding portion of an
antibody such as a single-chain antibody (scFv) or Domain
Antibodies (dAb) as described at www.domantis.com. Immunoassays for
drugs are described in U.S. Pat. Nos. 7,220,842, 5,677,132 and
5,618,926. Immunoassays for pesticides and pesticide degradation
products are described in U.S. Pat. No. 6,635,434. Drug and hormone
receptors may be native hormone receptors, or they may be
engineered species with similar binding properties as are known in
the art. (See U.S. Pat. Nos. 7,214,511, and 6,806,359, Rasmussen et
al, J. Biol. Chem., Vol. 276, Issue 7, 4717-4723, Feb. 16, 2001).
US Patent Publication 20050130208 discloses a cocaine-specific
aptamer that could be used as a binding portion of an
analyte-specific enzyme component or as an immobilized test
reagent. Other aptmers are listed on the database of the Ellington
research group at http://aptamer.icmb.utexas.edu/. Enzyme-aptamer
coupling has been described by Mir et al. "Different Strategies To
Develop an Electrochemical Thrombin Aptasensor." Electrochemical
Communication 8 (2006): 505-511.
[0046] The components of the reaction are combined in such a way
that they result in a difference in distribution symmetry of the
redox enzyme that is dependent on the presence or absence of
analyte in a sample. Various device formats can achieve this
result.
[0047] A. Sandwich Format
[0048] FIG. 3 shows two electrodes 31 and 32. Electrode 31 has
analyte-specific receptors 33 immobilized on the interior surface
thereof. Analyte 34 associates with the analyte specific receptors
33. Also present is an analyte-specific enzyme reagent 35 which
associates with analyte in such as way that the activity of the
enzyme is maintained in the bound and the unbound state, and a
redox substrate 36, 36'. Capture of the analyte-specific enzyme
reagent 35 occurs at the surface of electrode 31 when analyte 34 is
present because of the formation of receptor 33-analyte
34-analyte-specific enzyme species 35 sandwich. The activity of the
enzyme results in an increased concentration of one form of the
redox substrate (in the case of FIG. 3, of form 36) near electrode
31, and hence a detectable chemical potential gradient whose
magnitude is related to the amount of analyte develops. When the
analyte is not present, the sandwich does not form and thus no
detectable chemical potential gradient is produced.
[0049] B. Competition/Displacement Format
[0050] FIGS. 4 and 5 show a competition format for an assay device
in accordance with the invention. The sample cell is defined by two
electrodes 41, 42. Electrode 41 has analyte-receptors 43 disposed
thereon. The redox enzyme 45 comprises the enzyme coupled to an
analyte or an analyte mimetic. In the absence of analyte, as shown
in FIG. 4, redox enzyme 45 is coupled to analyte receptor 43,
resulting in an asymmetry in the distribution of the redox enzyme.
Additional redox enzyme 45' may be present in the bulk of the
sample depending on the amount of redox enzyme, analyte receptors
and the affinity of the analyte receptors for the redox enzyme. As
shown in FIG. 5, when analyte 44 is present, it competes with redox
enzyme 45 for the analyte receptor 43, resulting in the
displacement of at least some of the redox enzyme 45 from the
analyte receptors 43, creating a less asymmetric distribution of
the redox enzyme and thus the ability to determine analyte in a
sample.
[0051] C. Device Construction--Sample Cell with Facing
Electrodes
[0052] FIGS. 6A-C show a sample cell for a device in accordance
with the invention, constructed using facing electrodes. The device
is formed from a top layer 61 and bottom layer 62, each having a
conductive surface (61', 62') facing the interior of the device,
and an insulating spacer layer 63. Contacts 64 and 65 extend from
the top layer 61 and bottom layer 62 to allow contact with the
conductive surfaces for either potentiometric or amperometric
measurements. As shown in FIG. 6B, the conductive layer 63 has an
opening therein that together with the facing conductive surfaces
61' and 62' defines a sample cell. This sample cell is open to the
exterior for introduction of sample, for example through an opening
66 at the end device as shown in FIG. 6C. Various structures for
forming cells of this type, with multiple openings and/or vent
holes are known in the art for example as the particular design of
the cell is not critical to the practicing of the present
invention. Similarly, other designs for connectors are known, and
this also is not critical to the practicing of the present
invention.
[0053] In an embodiment of the device of the invention, one of the
conductive surfaces (61' or 62') has the analyte receptor
(immobilized test reagent) disposed thereon. The remainder of the
reagents for determination of analyte may be disposed within the
sample cell as a dried reagent layer 67, or may be added to the
sample prior to introduction to the sample cell.
[0054] The sample cell portion as shown in FIGS. 6A-C is generally
constructed as a single use, disposable component.
[0055] D. Device Construction--Side-by-Side Electrodes
[0056] In addition to facing electrodes as discussed above, the
first and electrodes may be disposed on the same surf-ace within
the sample cell. This configuration is referred to herein as
side-by-side electrodes. FIGS. 7A-C show non-limiting examples of
configurations that can be used for side-by-side configurations,
including parallel strips (FIG. 7A), interdigitated strips (7B) and
concentric rings (FIG. 7C). In each case, asymmetry in redox enzyme
distribution between one electrode 71 and the other electrode 72
results in a measurable chemical potential gradient between the two
electrodes.
[0057] The sample cell portions as shown in FIGS. 7A-C are
generally constructed as a single use, disposable component.
[0058] E. Device Construction--Communication of Results
[0059] In addition to the sample cell, the device of the invention
has a means for communicating the observed potential or current to
a user in a meaningful way indicative of the determination of
analyte. This can range from a simple qualitative result (analyte
present or not present), or to a specific numerical value for the
amount of analyte. The sophistication of the communication means
varies accordingly.
[0060] In one embodiment of the invention, to provide a qualitative
result, the device may have at a visible location a spot of a
material that changes color in response to the passage of current
or the application of the potential difference created by an
asymmetry above a threshold level. In this case, the device does
not need a separate meter portion, and may be simply a disposable
test strip with an indicator spot on the outside.
[0061] More commonly, the sample cell will be contained within a
disposable test strip that is inserted into a reusable meter. The
meter will contain the electronics for measuring the potential
difference or current at a defined period of time after
introduction of a sample or test strip, and for conversion of the
measured value to a displayed value. This conversion may make use
of a look-up table that converts specific value of current or
potential to values of analyte depending on the calibration values
for the specific device geometry and analyte.
[0062] FIG. 8 shows an external view of a meter in accordance with
the invention. As shown, the meter has a housing 81 having a
display 82 and one or more control interfaces 83 (for example power
buttons, or scroll wheels etc). The housing has a slot 84 for
receiving a test device. Internal to the housing 81 is circuitry
for applying a potential or current to the electrodes of the test
device when a sample is applied. This may be done following an
initialization signal from a user, or following an automated
detection of test device insertion and sample application.
[0063] Suitable circuits usable in the meter of the invention are
known in the art, for example from US Patent Publication No. US
2005-0265094 A1, which is incorporated herein by reference. One
such circuit is shown in FIG. 9. In FIG. 9, an ideal voltmeter 942
is provided which can measure the potential across the electrodes
941, 938. Switch 944 is provided which is opened when the potential
is to be measured or closed for measurement of current. When ope,
the cell 939 is "floating" as to at least one of its electrodes,
permitting a voltage measurement that is unaffected by signals at
the amplifier 935. The switch 944 may be a mechanical switch (e.g.
a relay) or an FET (field-effect transistor) switch, or a
solid-state switch. In a simple case the switch opens to an open
circuit; more generally it could open to a very high
resistance.
[0064] This circuit can be used to measure either a potential
difference or a current difference. As will be appreciated by
persons skilled in the art, other circuits, including much simpler
and more complicated circuits can be used to achieve application of
either or both of a potential difference or a current.
[0065] The circuit of FIG. 9 can also be used to apply a potential
difference to the test device of the invention. While, as discussed
above, such application of potential is not required to perform the
measurement of the invention, application of potential prior to the
measurement (particularly in a test device with facing electrode
construction) can result in a more rapid measurement time by
effectively driving analyte and/or analyte-binding enzyme component
towards the electrode with the immobilized reagent using what is in
essence electrophoresis, provided that the analyte or
analyte-binding enzyme component is charged under the conditions
(particularly pH) found within the test device. A step function or
a sine wave passing from negative to positive potential differences
could also effect something similar to washing in situ within the
test device.
VI. Advantages of the Invention
[0066] The present invention provides the ability to perform
binding assays for the detection of analyte with electrochemical
detection, without the need to apply an external potential or
current, and without the need for washing steps commonly employed
in sandwich immunoassay procedures. Because of this, the apparatus
used to perform the assay can be much simpler, easy to use, and
less expensive. In addition, the device components in an
electrochemical assay are more robust than those used for example
in optical measurements, facilitating the manufacture of the low
cost reusable meter for use with disposable test devices.
[0067] The present invention can make use of small samples of
blood, tears, saliva, or sweat, with minimal invasiveness to the
subject. (samples of less than 10 ul, preferably less than 1 ul are
particularly desirable for blood tests). Further, the present
invention can provide for very rapid test times. Common immunoassay
procedures take up to an hour or more. The small volume of the test
devices of the invention and the sensitivity of the test to small
differences in the distribution of the enzyme means that shorter
times (for example less the 30 minutes, more preferably less than
10 minutes, and most preferably less than 1 minute) are accessible
depending on the concentration of analyte to be measured and the
binding kinetics of the analyte with the binding elements of the
mobile and immobilized reagents.
[0068] Depending on the nature of the sample and the redox
substrate, it may be possible to use materials from the sample as
part of the reaction system. For example, endogenous glucose can be
used as a component of the redox substrate in a glucose
oxidase-based system when the sample is a blood sample.
EXAMPLES
[0069] In the following examples, the utility and operability of
the invention are demonstrated using a model system in which enzyme
is coated onto the surface of one of two electrodes in an electrode
pair. This model does not require the capture of the enzyme, and
therefore is useful for modeling the after-capture behavior with a
limited number of variables, although it does not provide a full
picture of the binding kinetics of an actual test system. Capture
of enzyme using reagents in other enzyme sandwich, competition and
displacement assays and the like is well known.
Example 1
[0070] An electrochemical cell test strip comprising two gold
electrodes separated by a double-sided adhesive layer was
constructed using the method described in US Patent Publication No.
US-2005-0258035-A1 which is incorporated herein by reference. An
total of 100 nL enzyme solution (27 mg/mL glucose oxidase in 100 mM
sodium citrate buffer pH 4.1) was dispensed onto the two electrodes
and allowed to dry. Test strips were made with different ratios of
electrode enzyme activity by apportioning the 100 nL enzyme
solution between the two electrodes, with the balance made up with
water. For example to make strips with 75% enzyme activity on
electrode 1 and 25% enzyme activity on electrode 2, 100 nL of a
mixture of 3 parts enzyme solution to 1 part water was dispensed
onto electrode 1, and 100 nL of a mixture of 1 part enzyme solution
to 3 parts water was dispensed onto electrode 2. A solution of 100
mM-beta-D-glucose and 100 mM potassium ferricyanide in water was
added to the electrochemical cell. The potential difference in open
circuit (FIG. 10), or current in short circuit (FIG. 11), was
recorded. It is noted that the period of time required to achieve
clear differences in the signal at the different levels of
asymmetry is short (less than 15 seconds) thus making the invention
suitable for rapid measurement.
[0071] FIG. 12 shows that peak potential difference attained
correlated with the ratio of enzyme activity dispensed on each
electrode. FIG. 13 shows that peak current attained correlated with
the ratio of enzyme activity dispensed on each electrode.
Example 2
[0072] By combining the determination of the ratio of enzyme
activity dispensed on each electrode (as in Example 1) with a
determination of total enzyme activity, the amount of enzyme (E1)
present at one electrode can be determined independent of the
amount (E2) present at the other electrode.
[0073] Varying amounts of enzyme were dispensed on 2 surfaces that
served as electrodes in a sandwich configuration as described in
Example 1. Ratio of enzyme activity present at the two electrodes
(R=E1/E2) was determined by measurement of the current flowing in a
short circuit configuration. Then the total enzyme activity
(E.sub.t=E1+E2) was determined by measuring the current flowing
with an applied potential difference. E1 and E2 were then
calculated from the determined values of R and E.sub.t.
[0074] Test strips containing a range of 0.25 microgram to 1
microgram of glucose oxidase were made, with the enzyme distributed
between the two electrodes such that R was between 1.5 and 19.
These strips were made by dispensing a solution of 0.25 microgram
to 1 microgram glucose oxidase in 100 nL of 100 mM sodium citrate
buffer pH 4.1 onto electrode 1, and a solution of 0 to 0.4
microgram glucose oxidase in 100 nL of 100 mM sodium citrate buffer
pH 4.1 onto electrode 2, then allowing the dispensed solutions to
dry.
[0075] A solution of 100 mM beta-D-glucose and 100 mM potassium
ferricyanide in water was added to the electrochemical cell and
current in short circuit was recorded for 5 seconds. Following
this, 300 mV was applied for 10 s. The current immediately prior to
5 s was averaged to give I.sub.0 and the current immediately prior
to 15 s was averaged to give I.sub.300 (i.e. the measured I at 5 s
is I.sub.0 and measured I at 15 s was designated as I.sub.300).
[0076] Measured I (both I.sub.0 and I.sub.300) for increasing
amounts of E.sub.t each with a variable ratio R, was recorded and
plotted.
[0077] FIG. 14 shows a schematic of known total enzyme E.sub.t on
the x-axis and measured I.sub.300 on the y-axis. The relationship
between E.sub.t and I.sub.300 can be established from this plot,
for example the data can be represented by the equation
I.sub.300=m.times.E.sub.t+n
where m and n are the slope and intercept of the line.
[0078] FIG. 15 shows a schematic plot of a known E.sub.t on x-axis
and measured I.sub.0 on the y-axis. As shown, a family of lines is
generated, one for each value of the ratio R. From FIG. 15, the
linear relationship of the I.sub.0 current vs E.sub.t is dependent
on R, ie
I.sub.0=f(E.sub.t, log R)
[0079] To further establish this function, we plotted known log (R)
vs slope (FIG. 16) for each line shown in FIG. 15. In addition, as
shown in FIG. 17 we plotted known log (R) and intercept for each
line in FIG. 15.
Thus the function f can further be defined as the relationship
I.sub.0=(a log R+b).times.E.sub.t+(c log R/d)
The parameters a and b can be calculated from the plots in FIG. 16
and the parameters c and d from FIG. 17. Once the parameters a, b,
c, d, m and n have been determined, E1 can be calculated as
follows: 1. Calculate E.sub.t from I.sub.300
E.sub.t=(I.sub.300-n)/m.
2. Calculate R from I.sub.0 and E.sub.t
log R=(I.sub.0-b.times.E.sub.t-d)/(a.times.E.sub.t+c)
R=10.sup.log R
3. Calculate E.sub.1 from E.sub.t and R
E.sub.1=(R.times.E.sub.t)/(1+R)
[0080] FIG. 18 shows calculated values of E.sub.1 versus actual
values of E.sub.1, for strips with varying amounts of enzyme on the
two electrodes. FIG. 18 indicates that our estimated values for the
enzyme dispensed on one electrode correlated well with the actual
amount that was dispensed, and were independent of the amount of
enzyme dispensed on the other electrode.
* * * * *
References