U.S. patent application number 13/437695 was filed with the patent office on 2013-01-03 for biosensor having improved hematocrit and oxygen biases.
This patent application is currently assigned to Abbott Diabetes Care Inc.. Invention is credited to Nigel J. Forrow, Shridhara Alva Karinka.
Application Number | 20130001101 13/437695 |
Document ID | / |
Family ID | 32106586 |
Filed Date | 2013-01-03 |
United States Patent
Application |
20130001101 |
Kind Code |
A1 |
Forrow; Nigel J. ; et
al. |
January 3, 2013 |
Biosensor Having Improved Hematocrit and Oxygen Biases
Abstract
The present invention provides reagent compositions, and analyte
measuring devices and methods that utilize the reagent
compositions.
Inventors: |
Forrow; Nigel J.; (Abingdon,
GB) ; Karinka; Shridhara Alva; (Pleasanton,
CA) |
Assignee: |
Abbott Diabetes Care Inc.
|
Family ID: |
32106586 |
Appl. No.: |
13/437695 |
Filed: |
April 2, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12815169 |
Jun 14, 2010 |
8163160 |
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13437695 |
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11420628 |
May 26, 2006 |
7754093 |
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12815169 |
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10278657 |
Oct 23, 2002 |
7501053 |
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11420628 |
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Current U.S.
Class: |
205/777.5 ;
204/403.14; 546/2 |
Current CPC
Class: |
C12Q 1/004 20130101;
C12Q 1/006 20130101 |
Class at
Publication: |
205/777.5 ;
204/403.14; 546/2 |
International
Class: |
G01N 27/327 20060101
G01N027/327; C07F 15/04 20060101 C07F015/04 |
Claims
1-65. (canceled)
66. A reagent composition, comprising: a mediator comprising a
phenanthroline quinone or a derivative thereof; a transitional
metal ion or heavier alkaline earth metal ion; and a counter
ion.
67. The reagent composition of claim 66, wherein the phenanthroline
quinone is a 1,10-phenanthroline quinone.
68. The reagent composition of claim 66, wherein the transition
metal ion is selected from the group consisting of nickel,
manganese, iron, cobalt, osmium and ruthenium.
69. The reagent composition of claim 68, wherein the transition
metal ion is nickel.
70. The reagent composition of claim 66, wherein the heavier earth
metal ion is selected from the group consisting of calcium,
strontium and barium.
71. The reagent composition of claim 66, wherein the counter anion
is a halide, a nitrate, a nitrite, a sulfate, a carbonate, a
phosphate, a thiocyanate, an acetate, a formate, a citrate, a
succinate, an oxalate, a tartrate, a benzoate, or an alkyl or
aromatic sulfonate.
72. The reagent composition of claim 71, wherein the halide is
chloride, bromide, fluoride, or iodide.
73. The reagent composition of claim 66, wherein the counter anion
is a tungstate, a molybdate, a ferricyanide, a nitroprusside, a
tetraphenylborate, an anionic dye or an anionic surfactant.
74. The reagent composition of claim 66, wherein the reagent
composition further comprises an enzyme.
75. The reagent composition of claim 74, wherein the enzyme is
NAD(P).sup.+-dependent dehydrogenase.
76. The reagent composition of claim 74, wherein the enzyme is
selected from the group consisting of alcohol dehydrogenase,
lactate dehydrogenase, glucose oxidase, glucose dehydrogenase,
cholesterol oxidase and 3-hydroxybutyrate dehydrogenase.
77. The reagent composition of claim 74, wherein the enzyme is
3-hydroxybutyrate dehydrogenase.
78. A biosensor comprising: an electrode support; a working
electrode disposed on the electrode support; a reference electrode
disposed on the electrode support; and a reagent composition
deposited over the working electrode and the reference electrode,
wherein the reagent composition comprises: an enzyme; a mediator
comprising a phenanthroline quinone or a derivative thereof; a
transitional metal ion or heavier alkaline earth metal ion; and a
counter ion.
79. The biosensor of claim 78, wherein the biosensor further
comprises a counter electrode.
80. The biosensor of claim 78, wherein the biosensor further
comprises a cover layer defining an enclosed space over the
electrodes, the cover layer having an aperture configured for
receiving a sample into the enclosed space.
81. The biosensor of claim 78, wherein the phenanthroline quinone
is a 1,10-phenanthroline quinone.
82. The biosensor of claim 78, wherein the enzyme is selected from
the group consisting of alcohol dehydrogenase, lactate
dehydrogenase, glucose oxidase, glucose dehydrogenase, cholesterol
oxidase and 3-hydroxybutyrate dehydrogenase.
83. The biosensor of claim 82, wherein the enzyme is
3-hydroxybutyrate dehydrogenase.
84. The biosensor of claim 78, wherein the transition metal ion is
selected from the group consisting of nickel, manganese, iron,
cobalt, osmium and ruthenium.
85. The biosensor of claim 84, wherein the transition metal ion is
nickel.
86. The biosensor of claim 78, wherein the heavier earth metal ion
is selected from the group consisting of calcium, strontium and
barium.
87. The biosensor of claim 78, wherein the counter anion is a
halide, a nitrate, a nitrite, a sulfate, a carbonate, a phosphate,
a thiocyanate, an acetate, a formate, a citrate, a succinate, an
oxalate, a tartrate, a benzoate, or an alkyl or aromatic
sulfonate.
88. The biosensor of claim 87, wherein the halide is chloride,
bromide, fluoride, or iodide.
89. The biosensor of claim 78, wherein the counter anion is a
tungstate, a molybdate, a ferricyanide, a nitroprusside, a
tetraphenylborate, an anionic dye or an anionic surfactant.
Description
CROSS-REFERENCE
[0001] This application is a continuation-in-part application of
Ser. No. 10/278,657, filed Oct. 23, 2002, which is incorporated
herein by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] All biosensors for determining the concentration of analytes
in a sample of blood suffer from hematocrit sensitivity to some
degree. The biosensor response decreases as the hematocrit of the
sample increases. There is no single reason for this decrease in
the signal, though some of the reasons include diminished diffusion
of the analyte in the sample and increased solution resistance. One
of the methods proposed for the elimination of hematocrit
sensitivity is to filter the red cells from the sample. The
membrane technology to filter red cells increases both the assay
time and measurement imprecision. Oxygen sensitivity has presented
a challenge. Biosensors employing the enzyme glucose dehydrogenase
are not expected to be oxygen sensitive. However, the
oxidation-reduction reactions of the mediator (or coenzyme) could
involve free radical intermediates. When these intermediates have
long lifetimes, molecular oxygen can quench them, thereby rendering
the chemistry sensitive to oxygen tension.
[0003] U.S. Pat. Nos. 5,708,247 and 5,951,836 describe a disposable
glucose test strip for use in a test meter of the type that
receives a disposable test strip and a sample of blood from a
patient and performs an electrochemical analysis. The working
formulation comprises a filler, an enzyme effective to oxidize
glucose, e.g., glucose oxidase, and a mediator effective to
transfer electrons from the enzyme. The working formulation is
printed over a conductive base layer to form a working electrode.
The filler, for example, a silica filler, is selected to have a
balance of hydrophobicity and hydrophilicity such that on drying it
forms a two-dimensional network on the surface of the conductive
base layer. The response of this test strip is claimed to be
temperature independent over relevant temperature ranges and is
substantially insensitive to the hematocrit of the patient.
[0004] In photometric biosensors, a membrane is typically used to
separate red cells from a sample of whole blood. The use of a
membrane increases the time of response. U.S. Pat. No. 6,271,045
describes a photometric biosensor that employs a correction method
to compensate for hematocrit sensitivity. The biosensor comprises a
support member that contains a spreading layer and a reagent layer,
and a capillary tube in communication with the support layer and
spreading layer for transporting a sample of body fluid thereto. A
capillary tube is provided on the support member whereby a fluid
containing an analyte to be tested is introduced into the tube and
flows through the tube to the spreading layer and contacts the
reagent layer. In order to compensate for hematocrit level in the
case of whole blood, additional sensors can be implemented so that
they inspect the capillary tube in the test device, one sensor at
the beginning of the capillary channel and one at the end. In this
biosensor, whole blood is applied to the capillary channel. The
entry flow of whole blood is timed as it moves between sensors. The
time that the blood takes to travel the length of the capillary
tube is an indication of the hematocrit of the blood. That
information is used to correct any shift in reflectance readings of
the instrument caused by the hematocrit level. It is also known
that the absorbance of hemoglobin can be measured, and the
measurement can be used to account for the sensitivity of the
measurement to hemoglobin.
[0005] The majority of electrochemical biosensors do not use
membrane technology; hence, electrochemical biosensors suffer from
hematocrit sensitivity. U.S. Pat. No. 6,284,125 describes a
biosensor insensitive to hematocrit, where red cells are separated
from plasma. U.S. Pat. No. 6,287,451 describes a biosensor that can
employ a method in which hematocrit level can be measured
electrochemically, and the corrected concentration of an analyte
can be determined from the measured concentration of the analyte
along with factors that depend on the sensitivity of the biosensor
to hematocrit level. The magnitude of the hematocrit sensitivity is
dependent on the type of biosensor and on the type of measurement.
For example, if the reaction is allowed to go to completion, the
lengthy reaction time allows for complete oxidation of the analyte
in the sample, thereby making the measurement less sensitive to
hematocrit.
[0006] U.S. Ser. No. 09/529,617, filed Jun. 7, 2000, incorporated
herein by reference, describes NAD.sup.+-dependent and
NAD(P).sup.+-dependent enzymes having substrates of clinical value,
such as glucose, D-3-hydroxybutyrate, lactate, ethanol, and
cholesterol. Amperometric electrodes for detection of these
substrates and other analytes can be designed by incorporating this
class of enzymes and establishing electrical communication with the
electrodes via the mediated oxidation of the reduced cofactors NADH
and NADPH. NAD.sup.+-dependent glucose dehydrogenase can be used as
the enzyme and 1,10-phenanthroline-5,6-dione isomer can be used as
the mediator. This combination shows hematocrit sensitivity and
oxygen sensitivity. The enzyme is not dependent on oxygen (oxygen
does not act as a co-substrate as it does with glucose oxidase) and
hence is expected to be insensitive to oxygen. However, the
mediator reaction appears to be slow and hence is affected by the
presence of oxygen. The mediation reaction involves free radical
intermediates. If the reaction is slow, the free radical
intermediates have longer half-life; hence, the probability of
being quenched by molecular oxygen is high. Accordingly, the enzyme
mediator combination shows oxygen dependency. The hematocrit bias
of 1,10-phenanthroline-5,6-dione mediator is not clearly
understood; however, it is speculated that the slow reaction rate
of the mediator is responsible for significant hematocrit
sensitivity. 4,7-Phenanthroline-5,6-dione does not exhibit as much
sensitivity to variations in hematocrit or oxygen as does
1,10-phenanthroline-5,6-dione. However, the structure of
1,10-phenanthroline-5,6-dione renders it easier to synthesize than
does the structure of 4,7-phenanthroline-5,6-dione. The starting
materials for the synthesis of 1,10-phenanthroline-5,6-dione are
much less expensive than are the starting materials for
4,7-phenanthroline-5,6-dione. Additionally, the reaction conditions
for the synthesis of 1,10-phenanthroline-5,6-dione are much less
severe than are the reaction conditions for
4,7-phenanthroline-5,6-dione. Accordingly, it would be desirable to
reduce the sensitivity of 1,10-phenanthroline-5,6-dione to
hematocrit sensitivity and oxygen sensitivity.
[0007] Glucose monitoring devices are calibrated at normal
hematocrit. In samples having a lower hematocrit, the biosensor
reads a higher than appropriate blood glucose level, and in samples
having a higher hematocrit, the biosensor reads a lower than
appropriate blood glucose level.
SUMMARY OF THE INVENTION
[0008] This invention involves a reagent composition that includes
a mediator, such as an isomer of phenanthroline quinone (PQ), such
as 1,10-phenanthroline-5,6-dione or a derivative thereof, a counter
anion, and at least one metal ion selected from the group
consisting of a transition metal ion, such as, for example, nickel,
manganese, iron, osmium, ruthenium, and the like, and heavier
alkaline earth metal ion, such as, for example, calcium, barium,
and the like. In certain embodiments, the reagent composition
further includes an enzyme dependent upon NAD(P).sup.+, such as,
for example, glucose dehydrogenase. In such embodiments, when used
with a biosensor, the reagent composition provides for improved
hematocrit bias and oxygen bias. In addition, the electrodes of
biosensors employing this reagent composition provide an accurate
clinical response over a hematocrit range that ranges from about
20% to about 70% and over an oxygen tension range that ranges from
about 1 kPa to about 20 kPa.
[0009] Although oxidation of glucose catalyzed by glucose
dehydrogenase is not oxygen sensitive, the mediator can be
sensitive to oxygen. The 1,10-phenanthroline-5,6-dione mediator has
the structural formula:
##STR00001##
The use of 1,10-phenanthroline-5,6-dione mediator in a glucose
biosensor is described in U.S. Ser. No. 09/529,617, filed June 7,
2000, now U.S. Pat. No. 6,736,957, the disclosure of which is
incorporated herein by reference in its entirety.
[0010] In one aspect, this invention provides a reagent composition
for use in an analyte sensing device, wherein the reagent
composition includes a mediator, wherein the mediator is
1,10-phenanthroline-5,6-dione or a derivative thereof, at least one
metal ion, and a counter anion. The metal ion may be a transition
metal ion or a heavier alkaline earth metal ion.
[0011] In another aspect, this invention provides a biosensor e.g.,
in the form of a strip, wherein the biosensor includes:
[0012] an electrode support having at least one electrode thereon;
and
[0013] a reagent composition adjacent to the at least one
electrode, the reagent composition including an enzyme, a
1,10-phenanthroline-5,6-dione or a derivative thereof as a
mediator, a metal ion, wherein the metal ion is a transition metal
ion or a heavier alkaline earth metal ion, and a counter anion.
[0014] In yet another aspect, this invention provides a biosensor,
e.g., in the form of a strip, wherein the biosensor has a working
electrode that includes a reagent composition that includes a
NAD(P).sup.+-dependent enzyme, 1,10-phenanthroline-5,6-dione or a
derivative thereof as a mediator, a metal ion, and a counter anion,
wherein the metal ion is a transition metal ion or a heavier
alkaline earth metal ion.
[0015] In one embodiment, the biosensor contains an electrode
arrangement that includes two electrodes. The biosensor
includes:
[0016] at least one electrode support;
[0017] a first electrode disposed on the electrode support, the
first electrode including a working area;
[0018] a second dual-purpose reference/counter electrode disposed
on the same electrode support as the first electrode or a different
electrode support, the dual-purpose reference/counter electrode
being spaced apart from the first electrode. As such, in certain
embodiments, wherein the first and second electrodes are on the
same electrode support, the electrodes are in a coplanar
configuration. In other embodiments, wherein the first and second
electrodes are on different electrode supports, the electrodes are
in a facing configuration. The term "facing electrodes" refers to a
configuration of the working and reference/counter electrodes in
which the working surface of the working electrode is disposed in
approximate opposition to a surface of the reference/counter
electrode.
[0019] In yet another embodiment, the biosensor contains an
electrode arrangement that includes three electrodes, wherein the
biosensor includes:
[0020] at least one electrode support;
[0021] a first electrode disposed on the electrode support, the
first electrode being a working electrode;
[0022] a second electrode disposed on the same electrode support as
the first electrode or a different electrode support, the second
electrode being a reference electrode;
[0023] a reagent composition deposited over the first electrode and
second electrode, wherein the reagent composition comprises an
enzyme, a mediator, a metal ion, and a counter anion, wherein the
mediator is 1,10-phenanthroline-5,6-dione or a derivative thereof,
and wherein the metal ion is a transition metal ion or a heavier
alkaline earth metal ion; and
[0024] optionally, a third electrode disposed on the same electrode
support as the first electrode or a different electrode support,
the third electrode being a counter electrode, the counter
electrode includes an electrically conductive material.
[0025] Embodiments of the invention described herein provide a
mediator that is substantially insensitive to hematocrit and
oxygen, thereby enabling the use of this mediator in hospital and
retail markets, where samples having extreme hematocrit ranges (20%
to 70%) and oxygen tensions (neonatal, venous, capillary and
arterial) are encountered. A biosensor in the form of a strip
employing this mediator can be used for numerous analytes, such as,
for example, glucose, ketone bodies, lactate, and alcohol, as well
as others.
[0026] Embodiments of the invention described herein exhibit
several advantages/benefits as compared with other biosensors that
are being used for similar purposes. These advantages/benefits may
include: [0027] 1. elimination of the requirement of a membrane or
any cross-linked network; [0028] 2. the ability to employ a kinetic
measurement, and consequently, the elimination of the requirement
to drive the reaction to completion, thereby eliminating the
hematocrit sensitivity; [0029] 3. the selection of an appropriate
reagent combination--enzyme/mediator/metal or enzyme/metal complex
of the mediator--is responsible for lower hematocrit sensitivity;
[0030] 4. the catalytic and electrochemical activity of the
mediator/metal combination or metal complex of the mediator is
responsible for oxygen and hematocrit insensitivity; [0031] 5.
improved performance is related to the choice of the combination of
mediator and metal ion; and [0032] 6. the ability to use a single
reagent composition for a working electrode and reference electrode
which dispenses with the need for a separate reference electrode
layer, thereby reducing the number of manufacturing steps.
[0033] Before the present invention is described, it is to be
understood that this invention is not limited to particular
embodiments described, as such may, of course, vary. It is also to
be understood that the terminology used herein is for the purpose
of describing particular embodiments only, and is not intended to
be limiting, since the scope of the present invention will be
limited only by the appended claims.
[0034] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limits of that range is also specifically disclosed. Each
smaller range between any stated value or intervening value in a
stated range and any other stated or intervening value in that
stated range is encompassed within the invention. The upper and
lower limits of these smaller ranges may independently be included
or excluded in the range, and each range where either, neither or
both limits are included in the smaller ranges is also encompassed
within the invention, subject to any specifically excluded limit in
the stated range. Where the stated range includes one or both of
the limits, ranges excluding either or both of those included
limits are also included in the invention.
[0035] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, some potential and exemplary methods and materials are
now described. All publications mentioned herein are incorporated
herein by reference to disclose and describe the methods and/or
materials in connection with which the publications are cited. It
is understood that the present disclosure supercedes any disclosure
of an incorporated publication to the extent there is a
contradiction.
[0036] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a strip" includes a plurality of such cells
and reference to "the compound" includes reference to one or more
compounds and equivalents thereof known to those skilled in the
art, and so forth.
[0037] It is further noted that the claims may be drafted to
exclude any optional element. As such, this statement is intended
to serve as antecedent basis for use of such exclusive terminology
as "solely", "only" and the like in connection with the recitation
of claim elements, or the use of a "negative" limitation.
[0038] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates which
may need to be independently confirmed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] The invention is best understood from the following detailed
description when read in conjunction with the accompanying
drawings. It is emphasized that, according to common practice, the
various features of the drawings are not to-scale. On the contrary,
the dimensions of the various features are arbitrarily expanded or
reduced for clarity. Included in the drawings are the following
figures:
[0040] FIG. 1 is a schematic diagram that illustrates a perspective
view of a biosensor strip having a working electrode and a
dual-purpose reference/counter electrode.
[0041] FIG. 2 is a schematic diagram that illustrates a perspective
view of a biosensor strip having a working electrode, a reference
electrode, and a counter electrode.
[0042] FIG. 3 is a graph showing electrochemical properties of
1,10-phenanthroline-5,6-dione in the absence of manganese chloride
(Curve 1) and in the presence of manganese chloride (Curve 2).
[0043] FIG. 4 is a graph showing the response of a biosensor as a
function of the concentration of glucose for three formulations
involving the mediator.
[0044] FIG. 5 is a graph showing the relative signals of biosensors
for of glucose (15 mM sample) as a function of hematocrit for three
formulations involving the mediator. The data are normalized to the
signal at 40% hematocrit.
[0045] FIG. 6 is a graph showing the relative oxygen sensitivities
of biosensors for three formulations involving the mediator. The
data are normalized to 7 kPa.
[0046] FIG. 7 is a comparison of the reagent film homogeneity of
ultra-soluble mediator [Ni(PQ).sub.3]Cl.sub.2 of the present
invention with the PQ mediator. Panel A is an image of a reagent
film derived from a solution containing 3% [Ni(PQ).sub.3]Cl.sub.2.
Panel B is an image of a reagent composition derived from a
suspension containing 3% PQ.
[0047] FIG. 8 is a graph showing the cyclic voltammetry properties
of PQ (solid line) and [Ni(PQ).sub.3]Cl.sub.2 (broken line) on
blank screen-printed carbon electrodes versus Ag/AgCl reference.
The graph shows that the [Ni(PQ).sub.3]Cl.sub.2 mediator of the
present invention has a similar oxidation potential (Eox) to the PQ
mediator.
[0048] FIG. 9 is a graph showing the electrode calibrations at 1,
2, and 3 second assay times with [Ni(PQ).sub.3]Cl.sub.2 and
D-3-hydroxybutyrate dehydrogenase (HBDH). FIG. 10 is a graph
showing the electrode calibrations at 1, 2, and 3 second assay
times with [Ni(PQ).sub.3]Cl.sub.2and glucose dehydrogenase with
nicotinamide adenine dinucleotide (GDH/NAD).
[0049] FIG. 11 is a graph showing the electrode calibrations at 1,
2, and 3 second assay times with [Ni(PQ).sub.3]Cl.sub.2and glucose
dehydrogenase with pyrroloquinolinequinone (GDH/PQQ).
DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION
[0050] In general, embodiments of the present invention provide
reagent compositions that include a mediator, such as an isomer of
phenanthroline quinone, 1,10-phenanthroline-5,6-dione or a
derivative thereof, a counter anion, and a metal ion. The metal ion
can be a transition metal ion or a heavier alkaline earth metal
ion. In addition, the present invention also provides biosensors
that utilize the reagent composition, as well as methods of using
the biosensors for detecting an analyte in a sample.
[0051] The structural formula of the mediator
1,10-phenanthroline-5, 6-dione is shown below:
##STR00002##
When the mediator is reduced by the enzyme, dimers or oligomers or
both are formed on account of intermolecular hydrogen bonding
between reduced 1,10-phenanthroline-5,6-dione molecules. These
oligomers are not soluble in the reaction medium and hence are not
readily regenerated for continued mediation. Intermolecular
hydrogen bonding of a dimer of reduced
1,10-phenanthroline-5,6-dione is shown below.
##STR00003##
The dimerization or oligomerization can be minimized in several
ways. The nitrogen atoms can be blocked by chemical modification. A
substituent, e.g., an alkyl group, can be added to one or both of
the nitrogen atoms in order to prevent the formation of hydrogen
bonds. Preventing the formation of hydrogen bonds also increases
the solubility of the mediator in both the oxidized and reduced
form. The methyl derivative of 1,10-phenanthroline-5,6-dione shows
increased solubility. The compound mediates the oxidation of NADH
in the biosensor strip, as described in U.S. Ser. No. 09/529,617,
filed Jun. 7, 2000, now U.S. Pat. No. 6,736,957, the disclosure of
which is incorporated herein by reference in its entirety. The
following structural formula illustrates mono-alkylated
1,10-phenanthroline-5,6-dione, where R represents an alkyl group,
such as, for example, --CH.sub.3 and X represents an anion such as
BF.sub.4.sup.-:
##STR00004##
Synthesis of alkylated compounds requires several steps. The alkyl
group is introduced after the 1,10-phenanthroline-5,6-dione is
formed. The oxidation-reduction properties of alkylated
1,10-phenanthroline-5,6-dione may not be dependent on metal ion
concentration, which would indicate that the alkylation process has
inhibited the formation of intermolecular hydrogen bonds.
[0052] The nitrogen atoms can also be blocked by the formation of a
complex having a coordination bond between a ligand and a metal
ion. Complexes can be formed prior to being used in a formulation
in the strip; alternatively, metal ions can simply be mixed with
the ink formulation that contains the mediator.
[0053] As used herein, the expression "transition metal" means
those elements of a metallic nature that have partially filled d or
f shells in any of their commonly occurring oxidation states. The
expression " heavier alkaline earth metals" means those elements of
a metallic nature that are in the IIA column of the periodic table
and that have an atomic number equal to or higher than 20.
[0054] The metal ions suitable for use in this invention include,
but are not limited to, nickel, manganese, zinc, calcium, iron,
ruthenium, cobalt, osmium, nickel, copper, rhenium, rhodium,
iridium, chromium, technetium, barium, strontium. The binding
efficiencies in these complexes are dependent on the particular
metal ion employed. For example, Mn (II) ions provide stronger
binding than do Mg (II) ions.
[0055] A representative metal complex of
1,10-phenanthroline-5,6-dione is:
##STR00005##
wherein M is selected from the group consisting of nickel,
manganese, iron, cobalt, osmium, ruthenium, calcium, strontium, and
barium.
[0056] In certain embodiments, the metal is nickel and the nickel
complex of 1,10-phenanthroline-5,6-dione is:
##STR00006##
[0057] The generic formula of the complex cation is shown below.
The ligands a, b, c, and d can represent two
1,10-phenanthroline-5,6-dione molecules or other monodentate
ligands, such as, for example, chloride, water, ammonia, or the
like, or multidentate ligands, such as, for example, bipyridyl or
the like, and M is selected from the group consisting of nickel,
manganese, iron, cobalt, osmium, ruthenium, calcium, strontium, and
barium.
##STR00007##
[0058] Counter anions suitable for use in this invention include,
but are not limited to, a halide, such as chloride, bromide,
fluoride, or iodide, a nitrate, a nitrite, a sulfate, a carbonate,
a phosphate, a thiocyanate, an acetate, a formate, a citrate, a
succinate, an oxalate, a tartrate, a benzoate, an alkyl or aromatic
sulfonate, a tungstate, a molybdate, a ferricyanide, a
nitroprusside, a tetraphenylborate, an anionic dye and an anionic
surfactant.
[0059] Biosensor strips suitable for this invention are illustrated
in FIGS. 1 and 2. Referring to FIG. 1, a biosensor strip 10
comprises an electrode support 12, e.g., an elongated strip of
polymeric material (e.g., polyvinyl chloride, polycarbonate,
polyester, or the like) supports three tracks 14a, 14b, and 14c of
electrically conductive ink, e.g., including carbon. These tracks
14a, 14b, and 14c determine the positions of electrical contacts
16a, 16b, and 16c, a dual-purpose reference/counter electrode 18, a
working electrode 20, and a trigger electrode 22. The electrical
contacts 16a, 16b, and 16c can be inserted into an appropriate
measurement device (not shown) for measurement of current.
[0060] Each of the elongated portions of the conductive tracks 14a,
14b, and 14c can optionally be overlaid with a track 24a, 24b, and
24c of conductive material, e.g., made of a mixture including
silver particles and silver chloride particles. The enlarged
exposed area 25 of track 24b overlies the dual-purpose
reference/counter electrode 18. A layer of a hydrophobic
electrically insulating material 26 further overlies the tracks
14a, 14b, and 14c. The positions of the dual-purpose
reference/counter electrode 18, the working electrode 20, the
trigger electrode 22, and the electrical contacts 16a, 16b, and 16c
are not covered by the layer of hydrophobic electrically insulating
material 26. This hydrophobic electrically insulating material 26
serves to prevent short circuits. Because this insulating material
is hydrophobic, it can cause the sample to be restricted to the
exposed electrodes. An exemplary insulating material is
commercially available "POLYPLAST" (Sericol Ltd., Broadstairs,
Kent, UK).
[0061] Optionally, a first layer of mesh 28, a second insulating
layer 30, a second layer of mesh 32, a third insulating layer 34,
and a tape 36 can overlay the hydrophobic insulating material. The
tape 36 includes a small aperture 38 to allow access of the applied
sample to the underlying layers of mesh 28 and 32. The second
insulating layer 30 and the third insulating layer 34 include
openings to allow access of the applied sample to the underlying
layers of mesh 28 and 32.
[0062] The working electrode 20 includes a layer of conductive
material containing a working area 20a. The working area 20a may be
formed from a reagent composition, which is added (e.g., printed)
on the layer of conductive material of the working electrode 20.
The reagent composition includes a mixture of an
oxidation-reduction mediator, a metal ion, a counter anion, an
enzyme, and, optionally, a conductive material.
[0063] The working area 20a may be formed from a printing ink that
includes the reagent composition described above, that includes a
mixture of an enzyme, an oxidation-reduction mediator, a counter
anion, a metal ion, and, optionally, a conductive material.
Alternatively, instead of an enzyme, the working area 20a can
contain a substrate that is catalytically reactive with an enzyme
to be assayed. The reagent composition is then applied to the
working electrode 20 and the dual-purpose reference/counter
electrode 18 as discrete areas of fixed length. In certain
embodiments, the conductive material includes particles of carbon
and the oxidation-reduction mediator comprises
1,10-phenanthroline-5,6-dione.
[0064] In other embodiments, the electrodes are formed on one or
more electrode supports by any suitable method including chemical
etching, laser ablation, photolithography, and the like. In
general, the electrode support is formed from an insulating
material, so that it will not provide an electrical connection
between the electrodes of the electrode set. Examples include
glass, ceramics and polymers. In certain embodiments, the electrode
substrate is a flexible polymer, such as a polyester or
polyimide.
[0065] For example, in the laser ablation process, the metallic
layer may be ablated into an electrode pattern. Furthermore the
patterned metallic layer may be coated or plated with additional
metal layers. For example, the metallic layer may be copper, which
is then ablated with a laser, into an electrode pattern;
subsequently, the copper may be plated with a titanium/tungsten
layer, and then a gold layer, to form the desired electrodes. In
certain embodiments, however, only a single layer of gold is used,
which is directly in contact with the electrode substrate. In such
embodiments, the reagent composition can be positioned adjacent to
the electrode(s).
[0066] In one such method, one or more channels are formed in the
substrate, for example by an embossing process using an embossing
die or roller. Other methods for forming the channels, such as the
use of a laser, or photolithography and etching of the substrate
can also be employed if desired.
[0067] The conductive material may contain pure metals or alloys,
or other materials which are metallic conductors. Examples include
aluminum, carbon (such as graphite), cobalt, copper, gallium, gold,
indium, iridium, iron, lead, magnesium, mercury (as an amalgam),
nickel, niobium, osmium, palladium, platinum, rhenium, rhodium,
selenium, silicon (such as highly doped polycrystalline silicon),
silver, tantalum, tin, titanium, tungsten, uranium, vanadium, zinc,
zirconium, mixtures thereof, and alloys or metallic compounds of
these elements. In certain embodiments, the conductive material
includes carbon, gold, platinum, palladium, iridium, or alloys of
these metals, since such noble metals and their alloys are
unreactive in biological systems.
[0068] The reagent composition includes an aqueous suspension of
the conductive material, a redox mediator, a metal ion, and a
counter anion. For the working electrode 20, the reagent
composition also includes an enzyme. For example, when the analyte
to be measured is glucose in blood, the enzyme is glucose
dehydrogenase, and the redox mediator is a
1,10-phenanthroline-5,6-dione. In the alternative, for the working
electrode 20, the printing ink can include a substrate in lieu of
an enzyme when the analyte to be measured is an enzyme.
[0069] In certain embodiments, the reagent composition can be
screen-printed. In such embodiments, the reagent composition can
further include a polysaccharide (e.g., a guar gum or an alginate),
a hydrolyzed gelatin, an enzyme stabilizer (e.g., glutamate or
trehalose), a film-forming polymer (e.g., a polyvinyl alcohol), a
conductive filler (e.g., carbon), a defoaming agent, a buffer, or a
combination of the foregoing.
[0070] The electrodes cannot be spaced so far apart that both the
working electrode 20 and the dual-purpose reference/counter
electrode 18 cannot be covered by the sample. In certain
embodiments, the length of the path to be traversed by the sample
(i.e., the sample path) is kept as short as possible in order to
minimize the volume of sample required. The maximum length of the
sample path can be as great as the length of the biosensor strip.
However, the corresponding increase in resistance of the sample
limits the length of the sample path to a distance that allows the
necessary response current to be generated. The resistance of the
sample is also influenced by the distance from the edge of the area
of the dual-purpose reference/counter electrode 18 to the edge of
the working area of the working electrode 20. Reducing this
distance by positioning the dual-purpose reference/counter
electrode 18 downstream from the working electrode 20 increases the
resistance of the sample. Positioning the electrodes contiguously
is conventional.
[0071] The trigger electrode 22 can be placed downstream of the
reference electrode. The trigger electrode 22 can be used to
determine when the sample has been applied to the strip, thereby
activating the assay protocol. See U.S. Ser. No. 09/529,617, filed
Jun. 7, 2000, now U.S. Pat. No. 6,736,957, the disclosure of which
is incorporated herein by reference in its entirety.
[0072] A biosensor strip 110 suitable for this invention is
illustrated in FIG. 2. Referring to FIG. 2, an electrode support
111, such as an elongated strip of polymeric material (e.g.,
polyvinyl chloride, polycarbonate, polyester, or the like) supports
three tracks 112a, 112b, and 112c of electrically conductive ink,
such as carbon. These tracks 112a, 112b, and 112c determine the
positions of electrical contacts 114a, 114b, and 114c, a reference
electrode 116, a working electrode 118, and a counter electrode
120. The electrical contacts 114a, 114b, and 114c are insertable
into an appropriate measurement device (not shown) for measurement
of current.
[0073] Each of the elongated portions of the conductive tracks
112a, 112b, and 112c can optionally be overlaid with a track 122a,
122b, and 122c of conductive material, for example made of a
mixture including silver particles and silver chloride particles.
The enlarged exposed area of track 122b overlies the reference
electrode 116. A layer of a hydrophobic electrically insulating
material 124 further overlies the tracks 112a, 112b, and 112c. The
positions of the reference electrode 116, the working electrode
118, the counter electrode 120, and the electrical contacts 114a,
114b, and 114c are not covered by the layer of hydrophobic
electrically insulating material 124. This hydrophobic electrically
insulating material 124 serves to prevent short circuits. The layer
of hydrophobic electrically insulating material 124 has an opening
126 formed therein. This opening 126 provides the boundary for the
reaction zone of the biosensor strip 110. Because this insulating
material is hydrophobic, it can cause the sample to be restricted
to the portions of the electrodes in the reaction zone. The working
electrode 118 comprises a layer of a non-reactive electrically
conductive material on which is deposited a layer 128 containing a
reagent composition for carrying out an oxidation-reduction
reaction. At least one layer of mesh 130 overlies the electrodes.
This layer of mesh 130 protects the printed components from
physical damage. The layer of mesh 130 also helps the sample to wet
the electrodes by reducing the surface tension of the sample,
thereby allowing it to spread evenly over the electrodes. A cover
132 encloses the surfaces of the electrodes that are not in contact
with the electrode support 111. This cover 132 is a liquid
impermeable membrane. The cover 132 includes a small aperture 134
to allow access of the applied sample to the underlying layer of
mesh 130.
[0074] The reagent composition 128 is deposited on that portion of
the electrically conductive material of the working electrode 118
where the oxidation-reduction reaction is to take place when a
sample is introduced to the biosensor strip 110. In such
embodiments, the reagent composition 128 can be applied to the
working electrode 118 as a discrete area having a fixed length.
Typical analytes of interest include, for example, glucose and
ketone bodies. Typical non-reactive electrically conductive
materials include, for example, carbon, platinum, palladium,
iridium, and gold. A semiconducting material such as indium doped
tin oxide can be used as the non-reactive electrically conductive
material. In certain embodiments, the reagent composition includes
a mixture of an oxidation-reduction mediator and an enzyme.
Alternatively, instead of an enzyme, the reagent composition can
contain a substrate that is catalytically reactive with an enzyme
to be assayed. In the biosensor strips of this invention, the
reagent(s) are applied in the form of a composition containing
particulate material and having binder(s), and, accordingly, does
not dissolve rapidly when subjected to the sample. In view of this
feature, the oxidation-reduction reaction will occur at the
interface of working electrode 118 and the sample. The glucose
molecules diffuse to the surface of the working electrode 118 and
react with the enzyme/mediator mixture.
[0075] In addition to being applied to the working electrode 118, a
layer of the reagent composition can be applied to any of the other
electrodes, such as the reference electrode when desired, as a
discrete area having a fixed length.
[0076] Other possible biosensor strip designs include those in
which the mesh layer 130 is eliminated, and the flow channel is of
such dimensions that the biosensor strip takes up a liquid sample
by capillary attraction. See U.S. Ser. No. 10/062,313, filed Feb.
1, 2002, incorporated herein by reference.
[0077] The mediator can be used for any NAD(P).sup.+ dependent
enzyme. Representative examples of these enzymes are set forth in
Table 1.
TABLE-US-00001 TABLE 1 E.C. (enzyme classification) Number Enzyme
name 1.1.1.1 Alcohol dehydrogenase 1.1.1.27 Lactate dehydrogenase
1.1.1.31 .beta.-hydroxybutyrate dehydrogenase 1.1.1.49
Glucose-6-phosphate dehydrogenase 1.1.1.47 Glucose dehydrogenase
1.2.1.46 Formaldehyde dehydrogenase 1.1.1.37 Malate dehydrogenase
1.1.1.209 3-hydroxysteroid dehydrogenase
Other enzyme systems that can be used with the mediator include,
but are not limited to, oxidases (glucose oxidase, cholesterol
oxidase, lactate oxidase). Formulations for screen-printing
reagents on an electrode comprise the components set forth in Table
2 and Table 3, where % means % by weight.
TABLE-US-00002 TABLE 2 (NAD)P.sup.+ dependent enzyme (such as
glucose 200 to 4000 units dehydrogenase) per gram Nicotinamide
adenine dinucleotide (NAD) 5 to 30% 1,10-phenanthroline-5,6-dione
0.1 to 1.5% Filler (such as carbon or silica) 10 to 30% Binder
(such as hydroxyethyl cellulose or guar 0.01 to 0.5% gum or
alginate) Protein stabilizer (such as trehalose or bovine 0.01 to
2% serum albumin) Metal ion 0.1 to 10% Buffers and other
electrolytes 1 to 10%
TABLE-US-00003 TABLE 3 (NAD)P.sup.+ dependent enzyme (such as
glucose 200 to 4000 units dehydrogenase) per gram Nicotinamide
adenine dinucleotide (NAD) 5 to 30% Metal complex of
1,10-phenanthroline-5,6-dione 0.1 to 1.5% Filler (such as carbon or
silica) 10 to 30% Binder (such as hydroxyethyl cellulose or guar
0.01 to 0.5% gum or alginate) Protein stabilizer (such as trehalose
or 0.01 to 15% bovine 0.01 to 15% serum albumin) Buffers and other
electrolytes 1 to 10%
[0078] The performance of biosensors for determining
electrochemical ketone bodies can also be enhanced with the use of
this chemistry. A typical formulation for determination of ketone
bodies is shown in Table 4.
TABLE-US-00004 TABLE 4 .beta.-hydroxybutyrate dehydrogenase 200 to
4000 units per gram Nicotinamide adenine dinucleotide (NAD) 5 to
30% 1,10-phenanthroline-5,6-dione 0.1 to 1.5% Filler (such as
carbon or silica) 10 to 30% Binder (such as hydroxyethyl cellulose
or guar 0.01 to 0.5% gum or alginate) Protein stabilizer (such as
trehalose or bovine 0.01 to 2% serum albumin) Metal ion 0.1 to 10%
Buffers and other electrolytes 1 to 10%
[0079] In general, NAD(P).sup.+-dependent enzymes react with
substrate according to the relationship
RH.sub.2+NAD(P).sup.+.fwdarw.R+NAD(P)H+H.sup.+
NAD(P)H is oxidized back to NAD(P).sup.+ by the mediator described
in this invention. The rate of this oxidation reaction is slower
than that of other isomers (1,7-phenanthroline-5,6-dione and
4,7-phenanthroline-5,6-dione). This slow reaction rate prevents
rapid regeneration of the coenzyme and hence makes it susceptible
to variation in hematocrit or oxygen in the sample. The mediator
will have higher probability of reacting with molecular oxygen and
hence become sensitive to oxygen. The diffusion of the mediator in
the sample is affected by the hematocrit variation and slow
reacting mediator will be more affected by restricted mobility
compared to a fast reacting mediator. The metal ions described
herein allow rapid regeneration of the coenzyme and hence makes it
less susceptible to variation in hematocrit or oxygen in the
sample.
EXAMPLES
[0080] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the present invention, and are
not intended to limit the scope of what the inventors regard as
their invention nor are they intended to represent that the
experiments below are all or the only experiments performed.
Efforts have been made to ensure accuracy with respect to numbers
used (e.g. amounts, temperature, etc.) but some experimental errors
and deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, molecular weight is weight average
molecular weight, temperature is in degrees Centigrade, and
pressure is at or near atmospheric.
Example 1
[0081] Metal ion is required for efficient mediation of NADH
oxidation by 1,10-phenanthroline-5,6-dione. In solution,
1,10-phenanthroline-5,6-dione does not show any electrochemical
oxidation at physiological pH conditions. In the presence of a
metal ion such as manganese, the mediator shows both oxidation and
reduction current. FIG. 3 shows the electrochemical properties of
1,10-phenanthroline-5,6-dione in the presence of manganese chloride
(Curve 2) and in the absence of manganese chloride (Curve 1).
[0082] The concentration of the metal ion required for the optimal
performance of the biosensor depends on the binding constant of the
metal and the 1,10-phenanthroline-5,6-dione. The efficiency of
complex formation and stability of the complex is dependent on the
metal ion. For example, only 10 mM manganese chloride is sufficient
to achieve the performance that is achieved by a 360 mM magnesium
chloride for 30 mM of 1,10-phenanthroline-5,6-dione in the
formulation. Ten (10) mM manganese chloride corresponds to a ratio
of one (1) metal ion to three (3) 1,10-phenanthroline-5,6-dione
molecules in the formulation that forms the metal complex. The
binding constant of Pb (II) with 1,10-phenanthroline-5,6-dione is
greater than the binding constant of Mn (II) or Mg (II) with
1,10-phenanthroline-5,6-dione; however, the enzyme is inactivated
by Pb (II). Mediation of NADH oxidation by
1,10-phenanthroline-5,6-dione in the presence of other transition
metal ions and heavier alkaline earth metal ions has been
demonstrated.
[0083] Transition metal ions and heavier alkaline earth metal ions
can also be used as complexes for the mediation of NADH oxidation.
The performance of the free ion Mn (II) mixed in the formulation is
identical to the performance of the complex that is formed before
it is added to the ink formulation.
[0084] The hematocrit and oxygen bias of formulations containing Mn
(II) are significantly improved compared to the formulations
containing Mg (II). FIG. 4 shows correlation of biosensor response
as a function of concentration of glucose for the three mediation
chemistries. FIG. 5 shows the relative signals of a 15 mM sample as
a function of hematocrit normalized to the signal at 40%
hematocrit. FIG. 6 shows oxygen sensitivities of the biosensors
with three chemistries normalized to 7 kPa. Similar hematocrit and
oxygen bias advantages are seen with the Fe (II) complex of
1,10-phenanthroline-5,6-dione. In other words, using a transition
metal ion or a heavier alkaline earth metal ion in the formulation
improves the electrochemical properties of the compound. Some of
the transition metal ions and heavier alkaline earth metal ions
show improved oxygen and hematocrit sensitivities as compared with
other transition metal ions and heavier alkaline earth metal
ions.
[0085] The complexes were either formed prior to use in the strip
or the metal ions were mixed with the ink. The metal ions used were
transition metal ions and heavier alkaline earth metal ions.
Example 2
Synthesis
[0086] Ultra-soluble [Ni(PQ).sub.3].sup.2+ salts were prepared by
adding solid PQ (3-fold excess) to a solution of the appropriate
nickel (II) salt. PQ was dissolves to form the Ni(II) complex. The
solution was filtered and then freeze-dried to give the solid
[Ni(PQ).sub.3].sup.2+ salt.
[0087] Slightly soluble [Ni(PQ).sub.3].sup.2+ salts were prepared
by adding the appropriate anion to a solution of an ultra-soluble
[Ni(PQ).sub.3].sup.2+ salt, e.g., [Ni(PQ).sub.3]Cl.sub.2 then
filtering the resulting precipitated product followed by
drying.
Aqueous Solubility
[0088] Solubility tests in water were performed on the new redox
mediators in comparison with the standard PQ mediators. Of the new
mediators prepared so far, the fluoride, chloride, bromide, iodide,
nitrate, sulfate and acetate salts of [Ni(PQ).sub.3].sup.2+ were
found to have aqueous solubility in excess of 20 mg/ml (2%)
compared to <4 mg/ml (<0.4%) for the standard PQ mediators.
As expected, the perchlorate, tetrafluroborate and
hexafluorophosphate salts of [Ni(PQ).sub.3].sup.2+ had very low
solubility in water (<0.5 mg/ml). A summary of the results is
provided in Table 5.
TABLE-US-00005 TABLE 5 Mediator Solubility in Water
[Ni(PQ).sub.3]Cl.sub.2 >400 mg/ml (>40%)
[Ni(PQ).sub.3]Br.sub.2 20 mg/ml (2%) [Ni(PQ).sub.3]I.sub.2 20 mg/ml
(2%) [Ni(PQ).sub.3]F.sub.2 >200 mg/ml (>20%)
[Ni(PQ).sub.3](NO.sub.3).sub.2 50 mg/ml (5%)
[Ni(PQ).sub.3](ClO.sub.4).sub.2 <0.5 mg/ml (<0.05%)
[Ni(PQ).sub.3](PF.sub.6).sub.2 <0.5 mg/ml (<0.05%)
[Ni(PQ).sub.3](BF.sub.4).sub.2 <0.5 mg/ml (<0.05%)
[Ni(PQ).sub.3](O.sub.2CCH.sub.3).sub.2 >500 mg/ml (>50%)
[Ni(PQ).sub.3]SO.sub.4 >500 mg/ml (>50%) PQ 2 mg/ml (0.2%)
[Mn(PQ).sub.3]Cl.sub.2 4 mg/ml (0.4%) [Fe(PQ).sub.3]Cl.sub.2
>200 mg/ml (>20%)
Reagent Film Homogeneity
[0089] A comparison of ultra-soluble mediator
[Ni(PQ).sub.3]Cl.sub.2 of the present invention with the standard
PQ mediator was also performed. FIG. 7 provides results of the
comparison of the reagent film homogeneity of ultra-soluble
mediator [Ni(PQ).sub.3]Cl.sub.2 of the present invention with the
PQ mediator. Panel A is an image of a reagent film derived from a
solution containing 3% [Ni(PQ).sub.3]Cl.sub.2. Panel B is an image
of a reagent composition derived from a suspension containing 3%
PQ. As shown in FIG. 7, the [Ni(PQ).sub.3]Cl.sub.2 mediator
provides a uniform reagent composition with no visible sign of
solid particles or crystals whereas the reagent composition derived
from the PQ mediator display a non-even distribution of solid
particles.
Electrochemistry
[0090] The electrochemistry properties the ultra-soluble mediator
[Ni(PQ).sub.3]Cl.sub.2 of the present invention and the standard PQ
mediator were also determined. The graph of FIG. 8 shows the cyclic
voltammetry properties of PQ (solid line) as compared to the
properties of [Ni(PQ).sub.3]Cl.sub.2 (broken line) on blank
screen-printed carbon electrodes versus Ag/AgCl reference. The
graph shows that the [Ni(PQ).sub.3]Cl.sub.2 mediator of the present
invention has a similar oxidation potential (Eox) to the standard
PQ mediator.
Examples of Electrode Calibrations with Different Enzymes
[0091] A stock aqueous reagent composition for use in analyte
sensing was prepared containing the following:
[0092] 3.4% [Ni(PQ).sub.3]Cl.sub.2,
[0093] 2% polymer,
[0094] 4.7% trehalose,
[0095] 0.7% MgCl.sub.2, and
[0096] 0.7% NAD (pH adjusted to 7).
[0097] Various enzymes were then added and the resulting solution
was used to coat gold electrodes which were dried for 3 min at
75.degree. C. Pre-formed capillary fill cells were then applied and
the strips trimmed to size. Testing was performed using the
appropriate substrate/analyte solutions in PBS. Test parameters
included the following: 0 sec delay, +200 mV applied potential (vs.
mediator redox couple--biamperometry), 100 Hz sampling time, manual
assay start. Electrode calibrations were performed at 1, 2, and 3
sec assay times. The working electrode reaction was based on:
[Ni(PQ).sub.3].sup.2++NADH=[Ni(PQH.sub.2).sub.3].sup.2++NAD
Then [Ni(PQH.sub.2).sub.3].sup.2+ is oxidized to
[Ni(PQ).sub.3].sup.2+ at the electrode. The reference electrode
reaction was based on the reduction of [Ni(PQ).sub.3].sup.2+ to
[Ni(PQH.sub.2).sub.3].sup.2+.
[0098] FIG. 9 shows the electrode calibrations at 1, 2, and 3
second assay times with [Ni(PQ).sub.3]Cl.sub.2and
D-3-hydroxybutyrate dehydrogenase (HBDH). FIG. 10 shows the
electrode calibrations at 1, 2, and 3 second assay times with
[Ni(PQ).sub.3]Cl.sub.2 and glucose dehydrogenase with nicotinamide
adenine dinucleotide (GDH/NAD). FIG. 11 shows the electrode
calibrations at 1, 2, and 3 second assay times with
[Ni(PQ).sub.3]Cl.sub.2 and glucose dehydrogenase with
pyrroloquinolinequinone (GDH/PQQ). Therefore, as provided in the
figures the reagent composition of the present invention is
suitable for use with a variety of different enzymes for detection
of an analyte.
[0099] The preceding merely illustrates the principles of the
invention. It will be appreciated that those skilled in the art
will be able to devise various arrangements which, although not
explicitly described or shown herein, embody the principles of the
invention and are included within its spirit and scope.
Furthermore, all examples and conditional language recited herein
are principally intended to aid the reader in understanding the
principles of the invention and the concepts contributed by the
inventors to furthering the art, and are to be construed as being
without limitation to such specifically recited examples and
conditions. Moreover, all statements herein reciting principles,
aspects, and embodiments of the invention as well as specific
examples thereof, are intended to encompass both structural and
functional equivalents thereof. Additionally, it is intended that
such equivalents include both currently known equivalents and
equivalents developed in the future, i.e., any elements developed
that perform the same function, regardless of structure. The scope
of the present invention, therefore, is not intended to be limited
to the exemplary embodiments shown and described herein. Rather,
the scope and spirit of present invention is embodied by the
appended claims.
* * * * *