U.S. patent application number 10/123414 was filed with the patent office on 2003-08-28 for apparatus for amperometric diagnostic anagnostic analysis.
This patent application is currently assigned to TALL OAK VENTURES. Invention is credited to Jordan, Colina L., Jordan, Joseph, Pottgen, Paul A., Szuminsky, Neil J., Talbott, Jonathan L..
Application Number | 20030159944 10/123414 |
Document ID | / |
Family ID | 23527629 |
Filed Date | 2003-08-28 |
United States Patent
Application |
20030159944 |
Kind Code |
A1 |
Pottgen, Paul A. ; et
al. |
August 28, 2003 |
Apparatus for amperometric diagnostic anagnostic analysis
Abstract
The present invention relates to a novel method and apparatus
for the amperometric determination of an analyte, and in
particular, to an apparatus for amperometric analysis utilizing a
novel disposable electroanalytical cell for the quantitative
determination of biologically important compounds from body
fluids.
Inventors: |
Pottgen, Paul A.; (Allison
Park, PA) ; Szuminsky, Neil J.; (Pittsburgh, PA)
; Talbott, Jonathan L.; (Freedom, PA) ; Jordan,
Joseph; (State College, PA) ; Jordan, Colina L.;
(Bellefonte, PA) |
Correspondence
Address: |
OBLON SPIVAK MCCLELLAND MAIER & NEUSTADT PC
FOURTH FLOOR
1755 JEFFERSON DAVIS HIGHWAY
ARLINGTON
VA
22202
US
|
Assignee: |
TALL OAK VENTURES
PITTSBURGH
PA
|
Family ID: |
23527629 |
Appl. No.: |
10/123414 |
Filed: |
April 17, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10123414 |
Apr 17, 2002 |
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09696253 |
Oct 26, 2000 |
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6413411 |
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09696253 |
Oct 26, 2000 |
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08386919 |
Feb 9, 1995 |
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6153069 |
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Current U.S.
Class: |
205/777.5 |
Current CPC
Class: |
G01N 27/3273 20130101;
Y10S 435/817 20130101 |
Class at
Publication: |
205/777.5 |
International
Class: |
G01N 027/327 |
Claims
We claim:
1. A sample cell for determining the concentration of a selected
compound in a sample aqueous fluid, comprising a first electrode
which acts as a working electrode. a second electrode of
substantially the same size as said first electrode and being made
of the same electrically conducting material as said first
electrode, said second electrode being operatively associated with
said first electrode, and at least one non-conducting layer member
having an opening therethrough, said layer member being disposed in
contact with at least one of said electrodes and said layer member
being sealed against at least one of said first and second
electrodes to form a known electrode area within said opening such
that said opening forms a well to receive said sample aqueous fluid
and to place said fluid in said known electrode area in contact
with said first electrode and second electrode, whereby
substantially the entire contents of said well is capable of being
substantially simultaneously subjected to a predetermined
reaction.
2. The sample cell of claim 1 wherein said first and second
electrodes comprise palladium.
3. The sample cell of claim 1 wherein said second electrode is a
reference electrode.
4. An apparatus for measuring compounds in a sample fluid,
comprising a) a housing having an access opening therethrough. b) a
sample cell receivable into said access opening of said housing,
said sample cell being composed of a first electrode which acts as
a working electrode, a second electrode which acts to fix the
system potential and provide opposing current flow with respect to
said first electrode, said second electrode being of substantially
the same size as said first electrode and being made of the same
electrically conducting material as said first electrode, said
second electrode being operatively associated with said first
electrode, at lease one non-conducting layer member having an
opening therethrough, said layer member being and said layer member
being sealed against at least one of said first and second
electrode to form a known electrode area within said opening such
that said opening forms a well to receive said sample fluid and to
place said fluid in said known electrode area in contact with said
first electrode and said second electrode, (c) means for applying
an electrical potential to said first electrode and said second
electrode, (d) means for creating an electrical circuit between
said first electrode and said second electrode through said sample,
(e) means for measuring Cottrell current through said sample and
(f) means for visually displaying results of said measurement.
5. The apparatus of claim 4 further including means for obtaining a
plurality of readings of current in said sample over a plurality of
measurement times, after said sample fluid has ben placed in said
well.
6. The cell of claim 1, said cell also including a reagent layer
positioned within said well created by said opening.
7. The cell of claim 6 wherein said reagent layer contains an
oxidant, a buffer and a binding agent.
8. The cell of claim 7 wherein said reagent layer is a layer of
said oxidant, buffer and binding agent coated onto a porous matrix
and said matrix is positioned within said cell.
9. The cell of claim 8 wherein said reagent layer is a mixture of
said oxidant, buffer and binding agent deposited directly into said
cell.
10. The cell of claim 7 wherein said oxidant is selected from the
group consisting of benzoquinone, ferricyanide, ferricinium, Cobalt
(III) orthophenanthroline, and Cobalt (III) dipyridyl.
11. The cell of claim 7 wherein said reagent layer also includes an
enzyme and said enzyme is an oxidoreductase.
12. The cell of claim 1 wherein said first electrode and said
second electrode comprises a nonconducting substrate to which said
electrically conducting material has been applied, wherein said
electrically conducting material is the same for each electrode and
selected from the group consisting of platinum, gold, palladium,
silver and carbon.
13. The cell of claim 1 wherein a first non-conducting layer member
has an opening therethrough and is positioned on said first
electrode, and said first electrode is positioned on said second
electrode, and said second electrode being positioned on a second
non-conducting layer member.
14. The cell of claim 1 wherein said first layer member includes a
plurality of notches therein exposing and defining an electrical
contact area on said first electrode, and said first electrode has
a notch therein to expose and define an electrical contact area on
said second electrode.
15. The cell of claim 1 wherein said first and second electrodes
are co-planarly positioned on a single substrate.
16. The cell of claim 1 wherein said second electrode has circular
opening therein and said opening of said layer member is concentric
with said opening of said layer member is concentric with said
openings of said second electrode, and said opening in said second
electrode is of smaller diameter than said opening of said layer
member whereby a circular functional electrode area is defined on
said second electrode, and said first electrode is positioned
beneath said second electrode such that said opening in said second
electrode exposes and defines a functional electrode area on said
first electrode.
17. The apparatus of claim 13 also comprising means for initiating
an electrical potential upon insertion of said sample aqueous fluid
to detect the presence of said sample aqueous fluid, and said
initiating means also having means for signaling microprocessor
means to commence a reaction timing sequence when the presence of
said sample aqueous fluid is detected, and means for removing said
potential during said reaction timing sequence.
18. A method of measuring the amount of a selected compound in body
fluids comprising: a) providing a measuring cell having at least a
first and second electrode of substantially the same size and made
of the same electrically conductive material, said cell further
containing an oxidant and a buffer, b) placing a sample of fluid to
be tested in said cell, c) reconstituting said oxidant and buffer
with said sample fluid to generate a predetermined reaction, d)
allowing said reaction to proceed substantially to completion, e)
applying a potential across said electrodes and sample, and f)
measuring the resulting Cottrell current to determine the
concentration of said selected compound present in said sample.
19. The method as set forth in claim 18 including providing as said
first electrode a working electrode and as said second electrode a
reference electrode.
20. The method of claim 18 including also providing in said cell
and enzyme as a catalyst and said enzyme is an oxidoreductase.
21. A method for measuring the amount of glucose in blood,
comprising a) providing a measuring cell having at least a f irst
and second electrode of substantially the same size and made of the
same electrically conductive material, said cell further containing
an oxidant, a buffer and an enzyme, b) placing a blood sample to be
tested in said cell, c) reconstituting said oxidant, buffer and
enzyme with said blood sample to generate a predetermined reaction,
d) essentially immediately applying a potential across said
electrodes and blood sample, and e) measuring the resultant
Cottrell current when the reaction has proceeded to completion to
determine the concentration of said glucose present in said blood
sample.
22. The method of claim 21 including selecting said oxidant from
the group consisting of benzoquinone, ferricyanide, ferricinium,
Cobalt (III) orthophenanthroline, and Cobalt (III) dipyridyl.
23. The method of claim 21 including selecting said oxidant from
the group consisting of benzoquinone, ferricyanide, ferricinium,
Cobalt (III) orthophenanthroline, and Cobalt (III) dipyridyl.
24. The method of claim 21 including providing as said first
electrode a working electrode and said second electrode a reference
electrode.
25. The method of claim 21 including adding as said enzyme, glucose
oxydase.
26. The method of claim 1 wherein in step b) said placing of the
blood sample to be tested in the cell generates a current and
initiates a timing sequence, and wherein the reaction of step d) is
allowed to proceed with an open circuit between said first and
second electrode.
27. The method of claim 18 wherein the measuring of said Cottrell
current includes obtaining a plurality of readings of current in
said cell over a plurality of measurement times after said
potential has been applied across said electrodes.
28. A method of measuring the amount of an analyte in a fluid
sample, comprising: a. adding the fluid sample to an
electrochemical cell having at least a first and second electrode
of substantially the same size and comprising the same electrically
conductive material, said electrochemical cell including an
electron transfer agent that will react in a reaction involving the
analyte, thereby forming a detectable species; b. incubating the
reaction involving analyte and electron transfer agent in an open
circuit for a specified period of time; c. applying a sufficient
potential difference between the electrodes of the electrochemical
cell, after the incubation step, to readily transfer at least one
electron between the detectable species and one of the electrodes,
thereby resulting in a Cottrell current; d. measuring the Cottrell
current; and e. correlating the measured Cottrell current to the
amount of analyte in the fluid sample.
29. The method of claim 28, wherein adding the fluid sample to the
electrochemical cell causes a sudden charging current, which
automatically initiates incubation step b) performed under open
circuit.
30. The method of claim 29, wherein the Cottrell current is
measured at a preset time following the incubation step.
31. The method of claim 28, wherein the electrochemical cell
further includes a catalyst in sufficient amount to catalyze the
reaction involving the analyte and the electron transfer agent.
32. The method of claim 31, wherein the catalyst is an enzyme.
33. The method of claim 32, wherein the analyte is glucose and the
enzyme is glucose oxidase.
34. The method of claim 28, wherein the electron transfer agent is
included in a reagent layer that is coated directly onto the
electrochemical cell or is incorporated into a supporting matrix
that is placed into the electrochemical cell.
35. The method of claim 34, wherein the supporting matrix is filter
paper, membrane, filter, woven fabric, or nonwoven fabric.
36. The method of claim 34, wherein the reagent layer further
includes a binder.
37. The method of claim 36, wherein the binder is gelatin,
carrageenan, methylcellulose, polyvinyl alcohol, or
polyvinylpyrrolidone.
38. The method of claim 37, wherein a dispersing, spreading, or
wicking layer overlays the reagent layer.
39. The method of claim 34, wherein adding the fluid sample to the
electrochemical cell causes a sudden charging current, which
automatically initiates incubation step b) performed under open
circuit.
40. The method of claim 39, wherein the Cottrell current is
measured at a preset time following the incubation step.
41. The method of claim 40, wherein the reagent layer further
includes an enzyme catalyst in sufficient amount to catalyze the
reaction involving the analyte and the electron transfer agent.
42. The method of claim 41, wherein the analyte is glucose in a
concentration from about 1 milligram glucose per deciliter of fluid
sample to about 1000 milligrams glucose per deciliter of fluid
sample, and the fluid sample is blood.
43. The method of claim 42, wherein the electron transfer agent is
ferricyanide, ferricinium, cobalt (III) orthophenanthroline, cobalt
(III) dipyridyl, or benzoquinone.
44. The method of claim 28, wherein the analyte is glucose, TSH,
T.sub.4, a hormone, a cardiac glycoside, an antiarrhythmic, an
antiepileptic, an antibiotic, cholesterol, or a non-therapeutic
drug.
45. The method of claim 28, wherein the measuring of said Cottrell
current includes obtaining a plurality of readings of current in
said cell over a plurality of measurement times after said
potential has been applied between said electrodes.
46. A method of measuring the amount of an analyte in a fluid
sample, comprising: a. adding the fluid sample to an
electrochemical cell that includes at least a first and second
electrode of substantially the same size and comprising the same
electrically conductive material, wherein the conductive material
is selected from the group consisting of palladium, platinum, gold,
silver, and carbon, an electron transfer agent, a first catalyst in
sufficient amount to catalyze a first reaction involving the
analyte, and a second catalyst in sufficient amount to catalyze a
second reaction involving a product of the first reaction and the
electron transfer agent, thereby forming a detectable species; b.
incubating the first and second reactions in an open circuit for a
specified period of time; c. applying a sufficient potential
difference between electrodes of the electrochemical cell, after
the incubation step, to readily transfer at least one electron
between the detectable species and one of the electrodes, thereby
resulting in a Cottrell current; d. measuring the Cottrell current;
and e. correlating the measured Cottrell current to the amount of
analyte in the fluid sample.
47. The method of claim 46, wherein adding the fluid sample to the
electrochemical cell causes a sudden charging current, which
automatically initiates incubation step b) performed under open
circuit.
48. The method of claim 47, wherein the Cottrell current is
measured at a preset time following the incubation step.
49. A method of measuring the amount of an analyte in a fluid
sample, comprising: a. adding the fluid sample to an
electrochemical cell that includes first and second electron
transfer agents, a first catalyst in sufficient amount to catalyze
a first reaction involving the analyte, a second catalyst in
sufficient amount to catalyze a second reaction involving a product
of the first reaction and the first electron transfer agent,
thereby forming an intermediate species that reacts with the second
electron transfer agent, thereby forming a detectable species; b.
incubating the reactions of step a) in an open circuit for a
specified period of time; c. applying a sufficient potential
difference between electrodes of the electrochemical cell having
substantially the same surface area and comprising the same
material, after the incubation step, to readily transfer at least
one electron between the detectable species and one of the
electrodes, thereby resulting in a Cottrell current; d. measuring
the Cottrell current; and e. correlating the measured Cottrell
current to the amount of analyte in the fluid sample.
50. The method of claim 49, wherein the measuring of said Cottrell
current includes obtaining a plurality of readings of current in
said cell over a plurality of measurement times after said
potential has been applied between said electrodes.
51. A method for measuring the amount of a selected compound in a
fluid sample, comprising: providing a measuring cell having at
least first and second electrodes of substantially the same size
and comprising the same electrically conductive material, for
contact with the fluid sample introduced into the cell, applying a
potential to the electrodes to detect the presence of the fluid
sample in the cell, placing the fluid sample into the cell,
removing the potential to the electrode after the fluid sample is
detected in the cell, selectively oxidizing the compound in the
fluid sample with an oxidized electron acceptor to produce an
oxidized form of the selected compound and a reduced electron
acceptor, and re-applying a potential across the cell electrodes
and measuring the resulting Cottrell current, said current being
proportional to the concentration of the reduced electron acceptor
and the selected compound in the fluid sample.
52. A method for measuring the amount of glucose in blood,
comprising: providing a measuring cell consisting of first and
second electrodes for contact with blood introduced into the cell,
said electrodes being of substantially the same size and comprising
the same electrically conductive material, applying a potential
across the electrodes, placing a volume of blood into the cell,
removing the potential across the electrodes after the volume of
blood is placed into the measuring cell, oxidizing the glucose in
the blood with an oxidized electron acceptor in the presence of
glucose oxidase to produce gluconic acid and a reduced electron
acceptor, re-applying a potential across the measuring cell
electrodes, and measuring the Cottrell current through the cell,
the Cottrell current being proportional to the glucose
concentration in the blood.
53. The method of claim 52, wherein placing the fluid sample into
the measuring cell causes a sudden charging current, which
automatically initiates removal of the potential from the
electrodes and performance of the selective oxidation of the
selected compound under open circuit.
54. The method of claim 53, wherein the Cottrell current is
measured at the preset time after re-application of a potential
across the measuring cell electrodes.
55. The method of claim 52, wherein placing the volume of blood
into the measuring cell causes a sudden charging current, which
automatically initiates removal of the potential across the
electrodes and performance of the oxidation of glucose in the blood
under open circuit.
56. The method of claim 55, wherein the Cottrell current is
measured at a preset time after re-application of a potential
across the measuring cell electrodes.
57. The method of claim 56, wherein the measuring of Cottrell
current includes obtaining a plurality of readings of current in
said cell after a plurality of pre-set measurement times after said
potential has been applied across said measuring cell
electrodes.
58. A device for analyzing an analyte, comprising: a. a first
electrical insulator; b. a pair of electrodes consisting of working
and second electrodes of substantially the same size, the
electrodes being made of the same electrically conducting materials
and being supported on the first electrical insulator; c. a second
electrical insulator, overlaying the first electrical insulator and
the electrodes and including a cutout portion that exposes
substantially equal surface areas of the working and second
electrodes; and d. a reagent substantially covering the exposed
electrode surfaces in the cutout portion and comprising the
oxidized form of a redox mediator, an enzyme, and a buffer, the
oxidized form of the redox mediator being of sufficient type to
receive at least one electron from a reaction involving enzyme,
analyte, and oxidized form of the redox mediator and being in
sufficient excess to insure that the diffusion limited
electrooxidation of the redox mediator at the working electrode
surface is the principle limiter of current flow through the device
and to resist a shift in potential between the electrodes, the
enzyme being of sufficient type and in sufficient amount to
catalyze the reaction involving enzyme, analyte, and oxidized form
of the redox mediator, and the buffer being unreactive with respect
to the reduced and oxidized form of the redox mediator and being of
sufficient type and in sufficient amount to provide and maintain a
pH at which the enzyme catalyzes the reaction involving enzyme,
analyte, and oxidized form of the redox mediator.
59. A reagent incorporated into a sample receiving portion of an
electrochemical device that measures an analyte and that has a pair
of electrodes consisting of working and second electrodes of
substantially the same size, the electrodes being made of the same
electrically conducting materials and having substantially equal
surface areas in the sample receiving portion, comprising: the
oxidized form of a redox mediator, an enzyme, and a buffer, the
oxidized form of the redox mediator being of sufficient type to
receive at least one electron from a reaction involving enzyme,
analyte, and oxidized form of the redox mediator and being in
sufficient amount to insure that current produced by diffusion
limited electrooxidation is limited by the oxidation of the reduced
form of the redox mediator at the working electrode surface, the
enzyme being of sufficient type and in sufficient amount to
catalyze the reaction involving enzyme, analyte, and oxidized form
of the redox mediator, and the buffer having a higher oxidation
potential than the reduced form of the redox mediator and being of
sufficient type and in sufficient amount to provide and maintain a
pH at which the enzyme, analyte, and oxidized form of the redox
mediator.
60. A reagent incorporated into a sample receiving portion of an
electrochemical device that measures an analyte and that has a pair
of electrodes consisting of working and second electrodes of
substantially the same size, the electrodes being made of the same
electrically conducting materials and having substantially equal
surface areas in the sample receiving portion, comprising: the
reduced form of a redox mediator, an enzyme, and buffer, the
reduced form of a redox mediator being of sufficient type to donate
at least one electron from a reaction involving enzyme, analyte,
and reduced form of the redox mediator and being in sufficient
amount to insure that current produced by diffusion limited
electroreduction is limited by the reduction of the oxidized form
of the redox mediator at the working electrode surface, the enzyme
being of sufficient type and in sufficient amount to catalyze the
reaction involving enzyme, analyte, and the reduced form of the
redox mediator, and the buffer having a lower reduction potential
than the oxidized form of the redox mediator and being of
sufficient type and in sufficient amount to provide and maintain a
pH at which the enzyme catalyzes the reaction involving enzyme,
analyte, and the reduced form of the redox mediator.
61. A method of determining the concentration of an analyte in a
fluid, comprising the steps of: a. contacting the fluid with a
reagent that covers substantially equal surface areas of first and
second electrodes and includes the oxidized form of a redox
mediator, an enzyme, and a buffer, the oxidized form of the redox
mediator being of sufficient type to receive at least one electron
from a reaction involving enzyme, analyte, and oxidized form of the
redox mediator and being in sufficient amount to insure that
current produced by diffusion limited electrooxidation is limited
by the oxidation of the reduced form of the redox mediator at the
working electrode surface, the enzyme being of sufficient type and
in sufficient amount to catalyze the reaction involving enzyme,
analyte, and the oxidized form of the redox mediator, and the
buffer having a higher oxidation potential than the reduced form of
the redox mediator and being of sufficient type and in sufficient
amount to provide and maintain a pH at which the enzyme catalyzes
the reaction involving enzyme, analyte, and the oxidized form of
the redox mediator; b. allowing the reaction involving the enzyme,
analyte, and the oxidized form of the redox mediator to go to
completion; c. subsequently applying a potential difference between
the electrodes sufficient to cause diffusion limited
electrooxidation of the reduced form of the redox mediator at the
surface of the first electrode; d. thereafter measuring the
resulting diffusion limited current; and e. correlating the current
measurement to the concentration of the analyte in the fluid.
62. The method of claim 61, wherein the reagent further includes a
supporting matrix material of sufficient type and in sufficient
amount to disperse the redox mediator in the reagent.
63. The method of claim 62, wherein the reagent further includes a
surfactant of sufficient type and in sufficient amount to wet the
fluid upon contact with the reagent.
64. The method of claim 63, wherein the analyte is glucose, the
oxidized form of the redox mediator is ferricyanide, the buffer is
phosphate, and the supporting matrix delays dissolution of the
reagent until said reagent has adsorbed said fluid.
65. The apparatus of claim 8, further including a wicking layer
positioned over said reagent layer and held in place with an
overlay tape.
66. The apparatus of claim 1, wherein the first and second
electrodes are spaced apart by a distance of at least about O.lmm
and at most about lcm.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a disposable
electro-analytical cell and a method and apparatus for
quantitatively determining the presence of biologically important
compounds such as glucose; hormones, therapeutic drugs and the like
from body fluids.
[0002] Although the present invention has broad applications, for
purposes of illustration of the invention specific emphasis will be
placed upon its application in quantitatively determining the
presence of a biologically important compound--glucose.
[0003] With Respect To Glucose
[0004] Diabetes, and specifically diabetes mellitus, is a metabolic
disease characterized by deficient insulin production by the
pancreas which results in abnormal levels of blood glucose.
Although this disease afflicts only approximately 4% of the
population in the United States, it is the third leading cause of
death following heart disease and cancer. With proper maintenance
of the patient's blood sugar through daily injection of insulin,
and strict control of dietary intake, the prognosis for diabetics
is excellent. The blood glucose levels must, however, be closely
followed in the patient either by clinical laboratory analysis or
by daily analyses which the patient can conduct using relatively
simple, non-technical, methods.
[0005] At present, current technology for monitoring blood glucose
is based upon visual or instrumental determination of color change
produced by enzymatic reactions on a dry reagent pad on a small
plastic strip. These calorimetric methods, which utilize the
natural oxidant of glucose to gluconic acid, specifically oxygen,
are based upon the reactions:
B-D-Glucose+O.sub.2+H.sub.2O.fwdarw.D-Gluconic
Acid+H.sub.2O.sub.2
H.sub.2O.sub.2+Reagent.fwdarw.H.sub.2O+color
[0006] Wherein glucose oxidase catalyzes the conversion of B-D
Glucose to D-Gluconic Acid. The hydrogen peroxide produced is
measured by reflectance spectroscopic methods by Acid. The hydrogen
peroxide produced is measured by reflectance spectroscopic methods
by its reaction with various dyes, in the presence of the enzyme
peroxidase, to produce a color that is monitored.
[0007] While relatively easy to use, these tests require consistent
user technique in order to yield reproducible results. For example,
these tests require the removal of blood from a reagent pad at
specified and critical time intervals. After the time interval,
excess blood must be removed by washing and blotting, or by
blotting alone, since the color measurement is taken at the top
surface of the reagent pad. Color development is either read
immediately or after a specified time interval.
[0008] These steps are dependent upon good and consistent operating
technique requiring strict attention to timing. Moreover, even
utilizing good operating technique, calorimetric methods for
determining glucose, for example, have been shown to have poor
precision and accuracy, particularly in the hypoglycemic range.
Furthermore, instruments used for the quantitative calorimetric
measurement vary widely in their calibration methods: some provide
no user calibration while others provide secondary standards.
[0009] Because of the general lack of precision and standardization
of the various methods and apparatus presently available to test
for biologically important compounds in body fluids, some
physicians are hesitant to use such equipment for monitoring levels
or dosage. They are particularly hesitant in recommending such
methods for use by the patients themselves. Accordingly, it is
desirable to have a method and apparatus which will permit not only
physician but patient self-testing of such compounds with greater
reliability.
[0010] The present invention addresses the concerns of the
physician by providing enzymatic amperometry methods and apparatus
for monitoring compounds within whole blood, serum, and other body
fluids. Enzymatic amperometry provides several advantages for
controlling or eliminating operator dependant techniques as well as
providing a greater linear dynamic range. A system based on this
type of method could address the concerns of the physician hesitant
to recommend self-testing for his patients.
[0011] Enzymatic amperometry methods have been applied to the
laboratory based measurement of a number of analytes including
glucose, blood urea nitrogen, and lactate. Traditionally the
electrodes in these systems consist of bulk metal wires, cylinders
or disks imbedded in an insulating material. The fabrication
process results in individualistic characteristics for each
electrode necessitating calibration of each sensor. These
electrodes are also too costly for disposable use, necessitating
meticulous attention to electrode maintenance for continued
reliable use. This maintenance is not likely to be performed
properly by untrained personnel (such as patients); therefore, to
be successful, an enzyme arnperometry method intended for
self-testing (or non-traditional site testing) must be based on a
disposable sensor that can be produced in a manner that allows it
to give reproducible output from sensor to sensor and at a cost
well below that of traditional electrodes.
[0012] The present invention addresses these requirements by
providing miniaturized disposable electroanalytic sample cells for
precise micro-aliquot sampling, a self-contained, automatic means
for measuring the electrochemical reduction of the sample, and a
method for using the cell and apparatus according to the present
invention.
[0013] The disposable cells according to the present invention are
preferably laminated layers of metallized plastic and nonconducting
material. The metallized layers provide the working and reference
electrodes, the areas of which are reproducibly defined by the
lamination process. An opening through these layers is designed to
provide the sample-containing area or cell for the precise
measurement of the sample. The insertion of the cell into the
apparatus according to the present invention, automatically
initiates the measurement cycle.
[0014] To better understand the process of measurement, a presently
preferred embodiment of the invention is described which involves a
two-step reaction sequence utilizing a chemical oxidation step
using other oxidants than oxygen, and an electrochemical reduction
step suitable for quantifying the reaction product of the first
step. One advantage to utilizing an oxidant other than dioxygen for
the direct determination of an analyte is that such other oxidants
may be prepositioned in the sensor in a large excess of the analyte
and thus ensure that the oxidant is not the limiting reagent (with
dioxygen, there is normally insufficient oxidant initially present
in the sensor solution for a quantitative conversion of the
analyte).
[0015] In the oxidation reaction, a sample containing glucose, for
example, is converted to gluconic acid and a reduction product of
the oxidant. This chemical oxidation reaction has been found to
precede to completion in the presence of an enzyme, glucose
oxidase, which is highly specific for the substrate B-D-glucose,
and catalyzes oxidations with single and double electron acceptors.
It has been found, however, that the oxidation process does not
proceed beyond the formation of gluconic acid, thus making this
reaction particularly suited for the electrochemical measurement of
glucose.
[0016] In a presently preferred embodiment, oxidations with one
electron acceptor using ferrocyanide, ferricinum, cobalt (III)
orthophenanthroline, and cobalt (III) dipyridyl are preferred.
Benzoquinone is a two electron acceptor which also provides
excellent electro-oxidation characteristics for amperometric
quantitation.
[0017] Amperometric determination of glucose, for example, in
accordance with the present invention utilizes Cottrell current
micro-chronoamperometry in which glucose plus an oxidized electron
acceptor produces gluconic acid and a reduced acceptor. This
determination involves a preceding chemical oxidation step
catalyzed by a bi-substrate bi-product enzymatic mechanism as will
become apparent throughout this specification.
[0018] In this method of quantification, the measurement of a
diffusion controlled current at one or more accurately specified
times (e.g., 5, 10, or 15 seconds) after the instant of application
of a potential has the applicable equation for amperometry at a
controlled potential (E=constant) of: 1 i COTTRELL at i > 0 = (
nFA ( Dt ) ) - 0.5 C analyte att = 0
[0019] which may also be expressed as:
i(t)=nFAC.sub.metabolite(D).sup.0.5(.pi.t).sup.-0.5
[0020] where i denotes current, nF is the number of coulombs per
mole, A is the area of the electrode, D is the diffusion
coefficient of the reduced form of the reagent, t is the preset
time at which the current is measured, and C is the concentration
of the metabolite. Measurements by the method according to the
present invention of the current due to the reoxidation of the
acceptors were found to be proportional to the glucose
concentration in the sample.
[0021] The method and apparatus of the present invention permit, in
preferred embodiments, direct measurements of blood glucose,
cholesterol and the like. Furthermore, the sample cell according to
the present invention, provides the testing of controlled volumes
of blood without premeasuring. Insertion of the sample cell into
the apparatus thus permits automatic functioning and timing of the
reaction allowing for patient self-testing with a very high degree
of precision and accuracy.
[0022] One of many of the presently preferred embodiments of the
invention for use in measuring B-D glucose is described in detail
to better understand the nature and scope of the invention. In
particular, the method and apparatus according to this embodiment
are designed to provide clinical self-monitoring of blood glucose
levels by a diabetic patient. The sample cell of the invention is
used to control the sampling volume and reaction media and acts as
the electrochemical sensor. In this described embodiment,
benzoquinone is used as the electron acceptor.
[0023] The basic chemical binary reaction utilized by the method
according to one preferred embodiment of the present invention
is:
B-D-glucose+Benzoquinone+H.sub.2O.fwdarw.Gluconic
Acid.fwdarw.Hydroquinone
Hydroquinone.fwdarw.benzoquinone-2e-+2H+.
[0024] The first reaction is an oxidation reaction which proceeds
to completion in the presence of the enzyme glucose oxidase.
Electrochemical oxidation takes place in the second part of the
reaction and provides the means for quantifying the amount of
hydroquinone produced in the oxidation reaction. This holds true
whether catalytic oxidation is conducted with two-electron
acceptors or one electron acceptor such as ferricyanide [wherein
the redox couple would be Fe(CN).sub.6.sup.-3/Fe(CN-
).sub.6.sup.-4], ferricinium, cobalt III orthophenanthroline and
cobalt (III) dipyridyl.
[0025] Catalytic oxidation by glucose oxidase is highly specific
for B-D-glucose, but is nonselective as to the oxidant. It has now
been discovered that the preferred oxidants described above have
sufficiently positive potentials to convert substantially all of
the B-D-glucose to gluconic acid. Furthermore, this system provides
a means by which amounts as small as 1 mg of glucose (in the
preferred embodiment) to 1000 mg of glucose can be measured per
deciliter of sample--results which have not previously been
obtained using other glucose self-testing systems.
[0026] The sensors containing the chemistry to perform the desired
determination, constructed in accordance with the present
invention, are used with a portable meter for self-testing systems.
In use, the sensor is inserted into the meter, which turns the
meter on and initiates a wait for the application of the sample.
The meter recognizes sample application by the sudden charging
current flow that occurs when the electrodes and the overlaying
reagent layer are initially wetted by the sample fluid. Once the
sample application is detected, the meter begins the reaction
incubation step (the length of which is chemistry dependent) to
allow the enzymatic reaction to reach completion. This period is on
the order of 15 to 90 seconds for glucose, with incubation times of
20 to 45 seconds preferred. Following the incubation period, the
instrument then imposes a known potential across the electrodes and
measures the resulting diffusion limited (i.e., Cottrell) current
at specific time points during the Cottrell current decay. Current
measurements can be made in the range of 2 to 30 seconds following
potential application with measurement times of 10 to 20 seconds
preferred. These current values are then used to calculate the
analyte concentration which is then displayed. The meter will then
wait for either the user to remove the sensor or for a
predetermined period before shutting itself down.
[0027] Due to the nature of the Cottrell current, it is possible to
develop a calibration curve at more than one time point following
application of the potential in order to verify that the
measurement is being accurately made. Results can then be
calculated at the different time points and compared. This is
illustrated schematically in FIGS. 11 and 12; which indicate
expected, or "normal" Cottrell curves, A, B, C, D, for various
glucose concentrations and an abnormal curve E, showing divergence
from expected curve D as indicated by the multiple current
readings. In a system that is operating correctly, the results
should agree within reasonable limits. The exact range of
acceptable difference between the expected and measured currents
depends on a number of compromises but would generally be in the
range of 1-10%. Results outside of the acceptable limits would
indicate some problem with the system. For instance, incomplete
wetting of the reagent (i.e., too small of a drop of blood) would
result in failure to follow the Cottrell curve decay and result in
a higher value being calculated at subsequent measurement points
than would have been expected for Cottrell current curve delay.
FIG. 13 represents a schematic circuit diagram which can be
employed in producing a preferred embodiment of the invention for
taking multiple current measurements.
[0028] The present invention provides for a measurement system that
eliminates several of the critical operator dependant variables
that adversely affect the accuracy and reliability and provides for
a greater dynamic range than other self-testing systems.
[0029] These and other advantages of the present invention will
become apparent from a perusal of the following detailed
description of one embodiment presently preferred for measuring
glucose and other analytes which is to be taken in conjunction with
the accompanying drawings in which like numerals indicate like
components and in which:
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIG. 1 is an exploded view of a portable testing apparatus
according to the present invention;
[0031] FIG. 2 is a plan view of the sampling cell of the present
invention;
[0032] FIG. 3 is an exploded view of the sample cell shown in FIG.
2;
[0033] FIG. 4 is an exploded view of another embodiment of a sample
cell according to the invention;
[0034] FIG. 5 is a plan view of the cell shown in FIG. 4;
[0035] FIG. 6 is a graph showing current as a function of glucose
concentration;
[0036] FIG. 7 is a graphical presentation of Cottrell current as a
function of glucose concentration; and
[0037] FIG. 8 is a presently preferred circuit diagram of an
electrical circuit for use in the apparatus shown in FIG. 1.
[0038] FIG. 9 is a preferred embodiment of the electrochemical cell
of the invention wherein the reference electrode area is greater
than the working electrode area.
[0039] FIG. 10 is a preferred embodiment of the invention wherein
the two electrodes are co- planar, equal size, and preferably the
same noble metal.
[0040] FIG. 11 is a graph showing multiple current measurements of
the present invention.
[0041] FIG. 12 is a graph correlating measured current to glucose
concentration for the curves of FIG. 11.
[0042] FIG. 13 is a schematic circuit diagram depicting a preferred
embodiment of the invention.
[0043] With specific reference to FIG. 1, a portable
electrochemical testing apparatus 10 is shown for use in patient
self-testing, such as, for example, for blood glucose levels.
Apparatus 10 comprises a front and back housing 11 and 12,
respectively, a front panel 13 and a circuit board 15. Front panel
13 includes graphic display panels 16 for providing information and
instructions to the patient, and direct read-out of the test
results. While a start button 18 is provided to initiate an
analysis, it is preferred that the system begin operation when a
sample cell 20 (FIG. 2) is inserted into the window 19 of the
apparatus.
[0044] With reference to FIGS. 2 and 3, sample cell 20 is a
metallized plastic substrate having a specifically-sized opening 21
which defines a volumetric well 21, when the cell is assembled, for
containing a reagent pad and the blood to be analyzed. Cell 20
comprises a first substrate 22 and a second substrate 23 which may
be preferably made from styrene or other substantially
non-conducting plastic, such as polyimide, polyethylene, etc.
Polyimide has proven particularly preferred because the metal
adheres well to it, and the electrodes may be more readily slitted
to the desired width. Polyimide sold under the trademark KAPTON,
and available from DuPont, has proven particularly
advantageous.
[0045] Positioned on second substrate 23 is reference electrode 24.
Reference electrode 24 may be preferably manufactured, for example,
by vapor depositing or "sputtering" the electrode onto a substrate
made from a material such as the polyimide Kapton. In one preferred
embodiment, reference electrode 24 is a silver--silver chloride
electrode. This electrode can be produced by first depositing a
silver chloride layer on a silver layer by either chemical or
electrochemical means before the substrate is used to construct the
cells. The silver chloride layer may even be generated in-situ on a
silver electrode when the reagent layer contains certain of the
oxidants, such as ferricyanide, and chloride as shown in the
following reactions:
Ag+Ox.fwdarw.Ag.sup.-+Red
Ag.sup.-+Cl.sup.-.fwdarw.AgCl
[0046] Alternatively the silver--silver chloride electrode can be
produced by depositing a layer of silver oxide (by reactive
sputtering) onto the silver film. The silver oxide layer is then
converted in-situ at the time of testing to silver chloride
according to the reaction:
Ag.sub.2O+H.sub.2O+2Cl.sup.-.fwdarw.2AgCl+2(OH).sup.-
[0047] when the sensor is wetted by the sample fluid and
reconstitutes the chloride containing reagent layer. The silver
electrode is thus coated with a layer containing silver
chloride.
[0048] The reference electrode may also be of the type generally
known as a "pseudo"reference electrode which relies upon the large
excess of the oxidizing species to establish a known potential at a
noble metal electrode. In a preferred embodiment, two electrodes of
the same noble metal or carbon are used, however one is generally
of greater surface area and is used as the reference electrode. The
large excess of the oxidized species and the larger surface area of
the reference resists a shift of the potential of the reference
electrode.
[0049] The primary requirement for the pseudo-reference (as is also
the case with traditional reference electrodes) is that it should
be able to supply the necessary current (in opposition to the
current flow at the working electrode) without significant shift in
its potential. Providing a larger surface area for the reference
electrode than for the working electrode is one way to accomplish
this. When high concentration of the oxidant are utilized and/or
the range of currents is kept relatively low, e.g., less than about
20 to 40 milliamps/cm.sup.2, with 0.1M ferricyanide as the oxidant,
it is possible to reduce the ratio of the reference to working
electrode to 1:1 or even less. The use of equal size electrodes
offers some advantage in terms of manufacture but with potentially
a limitation to the upper range.
[0050] In the case of same size (i.e., same surface area)
electrodes, a large excess of oxidized species (i.e., wherein the
oxidized form of the redox mediator (i.e., ferricyanide) is present
in the reagent layer in sufficient excess to insure that the
diffusion limited electrooxidation of the redox mediator at the
working electrode surface is the principal limiter of current flow
through the cell and resists a shift of the potential of the second
electrode (i.e., pseudo-reference) vis-a-vis the first (i.e.,
working) electrode.
[0051] In a highly preferred embodiment of the invention, the
two-electrode system uses same size, same metal electrodes.
Preferably noble metals, such as palladium are used. In this
palladium vs. palladium embodiment, a potential of +0.30 volts
applied between the electrodes has proven effective when
ferricyanide is the oxidant. Acceptable same-size palladium vs.
palladium coplanar-electrodes may be produced from metalized
plastic slitted by a high performance slitter such as Metlon Corp.,
133 Francis Avenue, Cranston, R.I. 02910.
[0052] In the pseudo-reference instance, care must be exercised in
the spacing of the electrodes. This is due to the fact that as the
second electrode functions to provide the balancing current, it is
actually producing the reduced form of the oxidant (redox
mediator). If the electrodes are spaced too closely together, the
reduced form produced at the second electrode can diffuse toward
the working electrode, where it would then be re-oxidized, adding
to the current flow due to the oxidation of the reduced form
produced by the enzymatic reaction. The net effect would be a
departure from the expected Cottrell current decay curve
illustrated in FIGS. 7 and 11.
[0053] This problem is potentially aggravated when the size of the
second electrode, vis-a-vis the first, is reduced, such as in the
case of same-size electrodes or when the second electrode is
smaller than the working electrode. This is due to the higher
effective concentration of reduced oxidant produced over the
surface of the second electrode. This can be shown by the following
analysis. The current flow at the two electrodes can be represented
by the Cottrell equation. For the first electrode, that is: 2 i t =
nFA 1 st DC 1 st Dt
[0054] At the second electrode that is: 3 i t = nFA 2 nd DC 2 nd
Dt
[0055] Since the two currents must be of the same magnitude (but
opposite sign), the following is true at any given time:
dC.sub.1stA.sub.1st=dC.sub.2ndA.sub.2nd
[0056] Where A.sub.1st is the area of the first (i.e., working)
electrode, C.sub.1st is the concentration of the reduced form of
oxidant in the solution in the reagent layer at time zero (the
instant the potential is applied), A.sub.2nd is the area of the
second (i.e., reference or counter) electrode, and C.sub.2nd is the
concentration of reduced form of the oxidant generated at or by the
second electrode at time zero (the instant the potential is
applied).
[0057] Since the working electrode is poised to control the current
flow, the second electrode acts to balance current flow. Thus, the
smaller the second electrode, the higher the concentration of the
reduced oxidant produced at the surface of that electrode; the
higher the concentration, the greater the diffusion gradient and
the greater the potential for the reduced oxidant produced at the
second electrode to diffuse towards the working electrode and cause
an undesired additional current flow, resulting in a departure from
expected Cottrell current flow for the system. First and second
electrodes 424, 426 must be spaced apart by a distance d (FIG. 10)
sufficient to avoid this departure from expected Cottrell current.
A distance d of at least 0.1 mm, preferably greater than 0.1 mm,
has proven effective at current flows of 0-50 microamps and about 5
mm.sup.2 working electrode area. The higher the expected current
density, the greater d must be in order to insure true Cottrell
measurement. Also, if measurement times exceed about 30 seconds, it
is necessary to increase the distance d. In a highly preferred
embodiment of the invention, d is about 1-2 mm, A.sub.1st and
A.sub.2nd are both about 5 mm.sup.2, current flow is about 0-100
microamps, with measurement time of less than 30 seconds.
[0058] The maximum spacing for d is dictated by the conductivity of
the solution, which effects to the actual voltage at the electrodes
(obviated in 3-electrode systems). Practical considerations,
however, dictate that the spacing d be as small as technically
feasible to mimnmize substrate, reagent, and sample size
requirements. Ideally, the preferred sample is the size of a drop
of blood of less than 20 microliters, which must bridge the
electrodes and wet the reagent layer. As a practical matter, the
spacing d should not exceed about 1 cm because of the difficulty of
bridging such a gap with a drop of blood of this size and
substantially instantaneously spreading that drop of blood across
the reagent layer. It is important that the sample, i.e., blood,
spread across the reagent layer substantially instantaneously
following application of the sample to the cell, in order to avoid
migration of reagents, or erosion of reagents from the site at
which the drop of blood was applied. The wicking layer is helpful
in this regard.
EXAMPLE 1
[0059] A two mm.sup.2 palladium indicator electrode was referenced
to a two mm.sup.2 Ag/Ag.sub.2O electrode and the electrodes were
spaced apart by a 2 mm gap. Measurements were taken from sample
cells including the reagents described herein, including an analyte
(i.e., glucose) the oxidized form of a redox mediator (i.e.,
ferricyanide), an enzyme (i.e., glucose oxidase), and a buffer
(i.e., phosphate). Preferably, in the case of same size, same noble
metal electrodes, the oxidized form of the redox mediator is
present in sufficient quantity to insure that the counter electrode
current, i.e., that produced at the reference electrode, is not
limiting as a result of conversion to the reduced form of the redox
mediator at the working electrode surface. The data for the
results, summarized in Table 1, were obtained 10 seconds after
current was applied.
[0060] For 5 millimolar ferrocyanide corresponding to 4 millimolar
glucose (72 mg/dl), an average current of 27.1 microamps was
obtained. The relative standard deviation was 5.3%. For 20
millimolar ferrocyanide, an average current of 102.9 was obtained
and the relative standard deviation was 3.9%. In this experiment,
the volumes tested were 50 microliters, although smaller volumes
such as 10 microliters could be employed if the area of the 2
mm.sup.2 electrode strips weree decreased.
[0061] A consentration series for ferrocyanide from 0 through 30
millimolar was similarly obtained. A plot of the currents at 10
seconds vs. ferrocyanide concentration indicated a linearly
increasing current with concentration through 25 millimolar
(approximately 360 mg/dl glucose). Similar results can be obtained
when both same-size electrodes are the same noble metal, such as
palladium.
[0062] The buffer used in the reagent layer is preferably
non-reactive with respect to the reduced and oxidized form of the
redox mediator (i.e., has a higher oxidation potential). Phosphate
buffer has proven effective in this regard, although other suitable
buffers would, of course, now be readily apparent to those of
ordinary skill in the art.
1 Corresponding Cottrell Current, Glucose microamps, at 10 seconds
Fe (CN).sub.6.sup.4- Concentration Concentration, after E is
applied millimolar mg/dl 0 0 0 27 5 72 58 10 144 75 15 216 103 20
288 142 25 360 136 30 432
[0063] Indicator or working electrode 26 can be either a strip of
platinum, gold, or palladium metallized plastic positioned on
reference electrode 24, or, alternately, as showned in FIG. 10, the
working electrode 426 and reference electrode 424 are laminated
between an upper 422 and lower 423 non-conducting (i.e.
electrically insulating) material, such as polyethylene or
polystyrene. Preferably, sample cell 20 is prepared by sandwiching
or laminating the electrodes between the substrate to form a
composite unit. Of course, other methods of applying the metal or
other electrically conducting material, such as, without
limitation, silk screening, vapor deposition, electrolysis,
adhesion, etc., may also be employed.
[0064] Of course, any combination of suitable electrode pairs may
be used. Names other than "working" vs. "reference" electrodes
which have been interchangeably used in electrochemical
applications include, "working" v.s. "counter" electrodes,
"excitation" v. "source" electrodes, or three electrode systems
having a "working" electrode, a "reference"electrode, and an
"auxiliary" electrode. In the case of a three-electrode system, the
auxiliary electrode completes the circuit, allowing current to flow
through the cell, while the reference electrode maintains a
constant interfacial potential difference regardless of the
current. In the two electrode scenario, the second electrode serves
both the finction of an auxiliary and reference electrode.
Regardless of the nomenclature employed, the electrochemistry of
interest in the present invention occurs at a first electrode
(i.e., working) and a second electrode (i.e., reference or counter)
acts to counter balance current flow (i.e., provide opposing
current flow to the first electrode) and fix the operating
potential of the system.
[0065] As shown in FIG. 2, first substrate 22 is of a slightly
shorter length so as to expose an end portion 27 of electrodes 24
and 26 and allow for electrical contact with the testing circuit
contained in the apparatus. In this embodiment, after a sample has
been positioned within well 21, cell 20 is pushed into window 19 of
the front panel to initiate testing. In this embodiment, a reagent
may be applied to well 21, or, preferably, a pad of dry reagent is
positioned therein and a sample (drop) of blood is placed into the
well 21 containing the reagent.
[0066] Referring to FIGS. 4-5, alternative embodiments of sample
cell 20 are shown. In FIG. 4, sample cell 120 is shown having first
122 and second 123 substrates. Reference electrode 124 and working
electrode 126 are laminated between substrates 122 and 123. Opening
121 is dimensioned to contain the sample for testing. End 130 (FIG.
5) is designed to be inserted into the apparatus, and electrical
contact is made with the respective electrodes through cut-outs 131
and 132 on the cell. Reference electrode 124 also includes cut out
133 to permit electrical contact with working electrode 126.
[0067] Referring to FIGS. 1 and 2, the sample cell according to the
present invention is positioned through window 19 (FIG. 1) to
initiate the testing procedure. Once inserted, a potential is
applied at portion 27 (FIG. 2) of the sample cell across electrodes
24 and 26 to detect the presence of the sample. Once the sample's
presence is detected, the potential is removed and the incubation
period initiated. Optionally during this period, a vibrator means
31 (FIG. 1) may be activated to provide agitation of the reagents
in order to enhance dissolution (an incubation period of 20 to 45
seconds is conveniently used for the determination of glucose and
no vibration is normally required). An electrical potential is next
applied at portion 27 of the sample cell to electrodes 24 and 26
and the current through the sample is measured and displayed on
display 16.
[0068] To fully take advantage of the above apparatus, the needed
chemistry for the self testing systems is incorporated into a dry
reagent layer that is positioned onto the disposable cell creating
a complete sensor for the intended analyte. The disposable
electrochemical cell is constructed by the lamination of metallized
plastics and nonconducting materials in such a way that there is a
precisely defined working electrode area. The reagent layer is
either directly coated onto the cell or preferably incorporated
(coated) into a supporting matrix such as filter paper, membrane
filter, woven fabric or non-woven fabric, which is then placed into
the cell, substantially covering the electrode surfaces exposed by
the cutout portion or window of the electrically insulating upper
laminate. When a supporting matrix is used, its pore size and void
volume can be adjusted to provide the desired precision and
mechanical support. In general, membrane filters or nonwoven
fabrics provide the best materials for the reagent layer support.
Pore sizes of 0.45 to 50 um and void volumes of 50-90% are
appropriate. The coating formulation generally includes a binder
such as gelatin, carrageenan, methylcellulose, polyvinyl alcohol,
polyvinylpyrrolidone, etc., that acts to delay the dissolution of
the reagents until the reagent layer has absorbed most of the fluid
from the sample. The concentration of the binder is generally on
the order of 0.1 to 10% with 1-4% preferred.
[0069] The reagent layer imbibes a fixed amount of the sample fluid
when it is applied to the surface of the layer thus eliminating any
need for premeasurement of sample volume. Furthermore, by virtue of
measuring current flow rather than reflected light, there is no
need to remove the blood from the surface of the reagent layer
prior to measurement as there is with reflectance spectroscopy
systems. While the fluid sample could be applied directly to the
surface of the reagent layer, to facilitate spread of blood across
the entire surface of the reagent layer the sensor preferably
includes a dispersing spreading or wicking layer. This layer,
generally a nonwoven fabric or adsorbent paper, is positioned over
the reagent layer and acts to rapidly distribute the blood over the
reagent layer. In some applications this dispersing layer could
incorporate additional reagents.
[0070] For glucose determination, cells utilizing the coplanar
design were constructed having the reagent layer containing the
following formulations: I
2 Glucose oxidase 600 units/ml Potassium Ferricyanide 0.4 M
Phosphate Buffer 0.1 M Potassium Chloride 05 M Gelatin 2.0 g/dl
[0071] This was produced by coating a membrane filter with a
solution of the above composition and air drying. The reagent layer
was then cut into strips that just fit the window opening of the
cells and these strips were placed over the electrodes exposed
within the windows. A wicking layer of a non-woven rayon fabric was
then placed over this reagent layer and held in place with an
overlay tape.
[0072] As will now be readily appreciated by those of ordinary
skill in the art, the enzyme (i.e., glucose oxidase) is sufficient
in type and amount to catalyze the reaction (i.e., receive at least
one electron from the reaction) involving the enzyme, the analyte
(i.e., glucose) and the oxidized form of the redox mediator (i.e.,
ferricyanide). Optionally, surfactant can also be used in the
reagent layer to promote wetting of the reagent by the sample
containing the analyte.
[0073] In order to prove the application of the technology
according to the present invention, a large number of examples were
run in aqueous solution at 25.degree. C. The electrolyte consisted
of a phosphate buffer of pH 6.8 which was about 0.1 molar total
phosphate and 0.5M potassium chloride reagent. The potentials are
referenced to a normal hydrogen electrode (NHE). In these tests it
was found that any potential between approximately +0.8 and 1.2
volt (vs NHE) is suitable for the quantification of hydroquinone
when benzoquinone is used as the oxidant. The limiting currents are
proportional to hydroquinone concentrations in the range between
0.0001M and 0.050M.
[0074] Detennination of glucose by Cottrell current (i.sub.t)
microchronoamperometry with the present method is created in the
reaction of hydroquinone to benzoquinone. Cottrell currents decay
with time in accordance with the equation:
.sup.i r.sup.t1/2=const
[0075] where t denotes time.
[0076] The main difference between these two techniques consists of
applying the appropriate controlled potential after the
glucose-benzoquinone reaction is complete and correlating glucose
concentrations with Cottrell currents measured at a fixed time
thereafter. The current-time readout is shown in FIG. 7.
Proportionality between glucose concentrations and Cottrell
currents (recorded at t=30 seconds after the application of
potential) is shown in FIG. 6.
[0077] It should be noted that Cottrell chronoamperometry of
metabolites needs the dual safeguards of enzymatic catalysis and
controlled potential electrolysis. Gluconic acid yields of 99.9+
percent were attained in the presence of glucose oxidase.
Concomitantly, equivalent amounts of benzoquinone were reduced to
hydroquinone, which was conveniently quantitated in quiescent
solutions, at stationary palladium thin film anodes or sample
cells.
[0078] The results of these many tests demonstrates the
microchronoamperometric methodology of the present invention and
its practicality for glucose self-monitoring by diabetics.
[0079] In a presently preferred embodiment of the invention
utilizing ferricyanide, a number of tests were run showing certain
improved operating capabilities.
[0080] Referring to FIG. 8, a schematic diagram of a preferred
circuit 15 for use in the apparatus 10 is shown. Circuit 15
includes a microprocessor and LCD panel 16. The working and
reference electrodes on the sample cell 20 make contact at contacts
W (working electrode) and R (reference electrode), respectively.
Voltage reference 41 is connected to battery 42 through analogue
power switchb 43. Current from electrodes W and R is converted to a
voltage by op amp 45. That voltage is converted into a digital
signal (frequency) by a voltage to frequency converter 46
electrically connected to the microprocessor 48. The microprocessor
48 controls the timing of the signals. Measurement of current flow
is converted by microprocessor 48 to equivalent glucose,
cholesterol or other substance concentrations. Other circuits
within the skills of a practiced engineer can now be utilized to
obtain the advantages of the present invention.
[0081] With regard to FIG. 9, cell 400 consists of coplanar first
(i.e., working) 426 and second (i.e., reference) 424 electrodes
laminated between an upper 422 and lower 423 nonconducting (i.e.,
electrically insulating) material. Lamination is on an adhesive
layer 425. The upper material 422 includes a die cut opening 428
which, along with the width of the working electrode material
defines the working electrode area and provides (with an
overlapping reagent layer not depicted) the sampling port of the
cell. As illustrated, this die-cut opening is rectangular or
square, which has proven advantageous from the standpoint of
reproduceability, as such openings are more readily centered over
the co-planar electrodes than circular openings. At one end of cell
400 is an open area 427 similar to end position 27 of FIG. 2. In
the case of same size (i.e., same surface area first and second
electrodes 426, 424 as illustrated in FIG. 10), the opening 428
insures that equal surface areas of the first and second electrodes
are exposed.
[0082] The efficiency of using the apparatus according to the
present invention to provide a means for in-home self testing by
patients such as diabetics (in the preferred embodiment) can be
seen in the following table in which the technology according to
the present invention is compared to four commercially available
units. As will be seen, the present invention is simpler, and in
this instance simplicity breeds consistency in results.
3 GLUCOSE SYSTEM COMPARISONS Present Steps 1 2 3 4 Invention Turn
Instrument On X X X X X Calibrate Instrument X X Finger Puncture X
X X X X Apply Blood X X X X X Initiate Timing X X X Sequence Blot X
X X Insert Strip to Read X X X X Read Results X X X X X Total Steps
Per 8 8 7 5 4 Testing Detection System RS' RS RS RS Polaro- graphic
Range (mg/dl) CV" 10-400 40-400 25-450 40-400 0-1000 Hypoglycemic
15% 15% 5% Euglycemic 10% 10% 3% Hyperglycermic 5% 5% 2%
Correlation 0.921 0.862 0.95 'RS - Reflectance Spectroscopy
"Coefficient of variation
[0083] Thus, while we have illustrated and described the preferred
embodiment of our invention, it is to be understood that this
invention is capable of variation and modification, and we
therefore do not wish or intend to be limited to the precise terms
set forth, but desire and intend to avail ourselves of such changes
and alterations which may be made for adapting the present
invention to various usages and conditions. Accordingly, such
changes and alterations are properly intended to be within the full
range of equivalents, and therefore within the purview, of the
following claims. The terms and expressions which have been
employed in the foregoing specifications are used therein as terms
of description and not of limitation, and thus there is no
intention, in the use of such terms and expressions, of excluding
equivalents of the features shown and described or portions
thereof, it being recognized that the scope of the invention is
defmed and limited only by the claims which follow.
[0084] Having thus described our invention and the manner and
process of making and using it in such full, clear, concise, and
exact terms so as to enable any person skilled in the art to which
it pertains, or to with which it is most nearly connected, to make
and use the same.
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