U.S. patent number RE36,268 [Application Number 08/679,312] was granted by the patent office on 1999-08-17 for method and apparatus for amperometric diagnostic analysis.
This patent grant is currently assigned to Boehringer Mannheim Corporation. Invention is credited to Joseph Jordan, deceased, Paul A. Pottgen, Neil J. Szuminsky, Jonathan L. Talbott.
United States Patent |
RE36,268 |
Szuminsky , et al. |
August 17, 1999 |
Method and apparatus for amperometric diagnostic 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: |
Szuminsky; Neil J. (Pittsburgh,
PA), Jordan, deceased; Joseph (late of State College,
PA), Pottgen; Paul A. (Allison Park, PA), Talbott;
Jonathan L. (Freedom, PA) |
Assignee: |
Boehringer Mannheim Corporation
(Indianapolis, IN)
|
Family
ID: |
27496781 |
Appl.
No.: |
08/679,312 |
Filed: |
July 12, 1996 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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176863 |
Dec 30, 1993 |
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322598 |
Mar 13, 1989 |
5128015 |
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168295 |
Mar 15, 1988 |
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Reissue of: |
745544 |
Aug 15, 1991 |
05108564 |
Apr 28, 1992 |
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Current U.S.
Class: |
205/777.5;
204/412; 205/782.5; 204/415; 435/287.1; 435/817; 435/289.1 |
Current CPC
Class: |
C12Q
1/004 (20130101) |
Current International
Class: |
C12Q
1/00 (20060101); G01N 33/487 (20060101); G01N
027/26 () |
Field of
Search: |
;204/403,415,412
;205/777.5,782.5 ;435/287.1,289.1,817 |
References Cited
[Referenced By]
U.S. Patent Documents
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WO |
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Other References
Van Nostrand Reinhold Encyclopedia of Chemistry; pp. 149-150 (4th
edition 1984) no month available. .
Biochemica Information; pp. 18, 19, 27 and 28 (J. Keesey, ed.,
Boehringer Mannheim Biochemicals, 1987) no month available. .
Laboratory Techniques in Electroanalytical Chemistry; pp. 51-64 and
124 (Kissinger and Heineman, eds., 1984) no month available. .
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Analysis", Feb. 1988, vol. 37, pp. 5-12, Microchemical Journal.
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Pennsylvania State University (Thesis for Doctor of Philosophy in
Chemistry) no month available..
|
Primary Examiner: Bell; Bruce F.
Attorney, Agent or Firm: Young; D. Michael
Parent Case Text
The present application .Iadd.is a continuation of Ser. No.
08/176,863 filed Dec. 30, 1993, now abandoned; which is a Re-issue
of Ser. No. 07/745,544 filed Aug. 15, 1991, now U.S. Pat. No.
5,108,564 which .Iaddend.is a division of application Ser. No.
07/322,598, filed Mar. 13, 1989, which is a continuation-in-part of
our earlier filed application, U.S. Ser. No. 168,295, filed Mar.
15, 1988.Iadd., now abandoned.Iaddend..
Claims
We claim:
1. 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 and said cell 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.
2. A method as set forth in claim 1, wherein the compound is
selected from the group consisting of glucose, cholesterol, TSH,
T4, hormones, antiarrhythmics, antiepileptics and nontherapeutic
drugs.
3. A method as set forth in claim 1, wherein the oxidant is a
material selected from the group consisting of benzoquinone,
ferricyanide, ferricinium, .Iadd.orthophenanthroline .Iaddend.and
Cobalt (III) dipyridyl.
4. The method as set forth in claim 1 including providing
as said first electrode a working electrode and as said second
electrode a reference electrode.
5. The method of claim 1 including also providing in said cell
.[.and.]. .Iadd.an .Iaddend.enzyme as a catalyst and said enzyme is
an oxidoreductase.
6. The method of claim 1 including selecting said buffer from the
group consisting of phosphate, TRIS, MOPS, MES, HEPES, Tricine,
Bicine, ACES, CAPS and TAPS. .[.7. A method for measuring the
amount of glucose in blood, comprising
a) providing a measuring cell having at least a first and second
electrode and said cell 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
blood sample..]..[.8. The method of claim 7 including selecting
said oxidant from the group consisting of benzoquinone,
ferricyanide, ferricinium, Cobalt (III) orthophenantroline, and
Cobalt (III) dipyridyl..]..[.9. The method of claim 7 including
providing as said first electrode a working electrode and said
second electrode a reference electrode..]..[.10. The method of
claim 7 including adding as said enzyme,
glucose oxydase..].11. The method of claim 1 wherein .Iadd.the
sample of fluid is blood and .Iaddend.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.]. .Iadd.electrodes.Iaddend.. .Iadd.12. A
method of measuring the amount of an analyte in a blood sample,
comprising:
a) adding the blood sample to an electrochemical cell that includes
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 until the reaction has substantially
completed;
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 blood sample..Iaddend..Iadd.13. The method of claim 12, wherein
adding the blood sample to the electrochemical cell causes a sudden
charging current, which automatically initiates incubation step b)
performed under open circuit..Iaddend..Iadd.14. The method of claim
13, wherein the Cottrell current is measured at a preset time
following the incubation step..Iaddend..Iadd.15. The method of
claim 12, wherein the electrochemical cell further includes a
catalyst in sufficient amount to catalyze the reaction involving
the analyte and the electron transfer agent..Iaddend..Iadd.16. The
method of claim 15, wherein the catalyst is an
enzyme..Iaddend..Iadd.17. The method of claim 16, wherein the
analyte is glucose and the enzyme is glucose
oxidase..Iaddend..Iadd.18. The method of claim 12, 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..Iaddend..Iadd.19. The method of claim 18, wherein the
supporting matrix is filter paper, membrane filter, woven fabric,
or nonwoven fabric..Iaddend..Iadd.20. The method of claim 18,
wherein the reagent layer further includes a
binder..Iaddend..Iadd.21. The method of claim 20, wherein the
binder is gelatin, carrageenan, methylcellulose, polyvinyl alcohol,
or polyvinylpyrrolidone..Iaddend..Iadd.22. The method of claim 21,
wherein a dispersing, spreading, or wicking layer overlays the
reagent layer..Iaddend..Iadd.23. The method of claim 18, wherein
adding the blood sample to the electrochemical cell causes a sudden
charging current, which automatically initiates incubation step b)
performed under open circuit..Iaddend..Iadd.24. The method of claim
23, wherein the Cottrell current is measured at a preset time
following the incubation step..Iaddend..Iadd.25. The method of
claim 24, wherein the reagent layer further includes an enzyme
catalyst in sufficient amount to catalyze the reaction involving
the analyte and the electron transfer
agent..Iaddend..Iadd.26. The method of claim 25, wherein the
analyte is glucose in a concentration from about 1 milligram
glucose per deciliter of blood sample to about 1000 milligrams
glucose per deciliter of blood sample, and the fluid sample is
blood..Iaddend..Iadd.27. The method of claim 26, wherein the
electron transfer agent is ferricyanide, ferricinium, cobalt (III)
orthophenanthroline, cobalt (III) dipyridyl, or
benzoquinone..Iaddend..Iadd.28. The method of claim 12, 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..Iaddend..Iadd.29. The method of claim 25,
wherein the analyte is glucose in a concentration from about 1
milligram glucose per deciliter of blood sample to about 1000
milligrams glucose per deciliter of blood sample, the incubation
period is from about 15 seconds to about 160 seconds, and current
measurements are made in the range from about 2 seconds to about 30
seconds following the incubation step..Iaddend..Iadd.30. A method
of measuring the amount of an analyte in a blood sample,
comprising:
a) adding the blood sample to an electrochemical cell that
includes
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
until the reactions have substantially completed;
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 blood sample..Iaddend..Iadd.31. The method of claim 30, wherein
adding the blood sample to the electrochemical cell causes a sudden
charging current, which automatically initiates incubation step b)
performed under open circuit..Iaddend..Iadd.32. The method of claim
31, wherein the Cottrell current is measured at a preset time
following the incubation step..Iaddend..Iadd.33. A method of
measuring the amount of cholesterol in a blood sample,
comprising:
a) adding the blood sample to an electrochemical cell that
includes
an electron transfer agent,
cholesterol esterase in sufficient amount to catalyze the
hydrolysis of cholesterol esters in the blood sample, thereby
forming cholesterol,
cholesterol oxidase in sufficient amount to catalyze a reaction
involving cholesterol and the electron transfer agent, thereby
forming a detectable species;
b) incubating the reactions of step a) in an open circuit until
those reactions have substantially completed;
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
cholesterol in the blood sample..Iaddend..Iadd.34. The method of
claim 33, wherein adding the blood sample to the electrochemical
cell causes a sudden charging current, which automatically
initiates incubation step b) performed under open
circuit..Iaddend..Iadd.35. The method of claim 34, wherein the
Cottrell current is measured at a preset time following the
incubation step..Iaddend..Iadd.36. The method of claim 35, wherein
the electron transfer agent is ferricyanide or
benzoquinone..Iaddend..Iadd. A method of measuring the amount of an
analyte in a blood sample, comprising:
a) adding the blood 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 until
those reactions have substantially completed;
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 blood sample..Iaddend..Iadd.38. A method of
measuring the amount of cholesterol in a blood sample,
comprising:
a) adding the blood sample to an electrochemical cell that
includes
first and second electron transfer agents,
cholesterol esterase in sufficient amount to catalyze the
hydrolysis of cholesterol esters in the blood sample, thereby
forming cholesterol,
cholesterol oxidase in sufficient amount to catalyze a reaction
involving cholesterol 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 until
those reactions have substantially completed;
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
cholesterol
in the blood sample..Iaddend..Iadd.39. The method of claim 38,
wherein adding the blood sample to the electrochemical cell causes
a sudden charging current, which automatically initiates incubation
step b) performed under open circuit..Iaddend..Iadd.40. The method
of claim 39, wherein the Cottrell current is measured at a preset
time following the incubation step..Iaddend..Iadd.41. The method of
claim 40, wherein the first electron transfer agent is
benzoquinone, phenazine ethosulfate, phenazine methosulfate,
tetramethylbenzidine, a derivative of benzoquinone, naphthoquinone,
a derivative of naphthoquinone, anthraquinone, a derivative of
anthraquinone, catechol, phenylenediamine,
tetramethylphenylenediamine, or a derivative of
phenylenediamine..Iaddend..Iadd.42. The method of claim 41, wherein
the
second electron transfer agent is ferricyanide..Iaddend..Iadd.43. A
method for measuring the amount of a selected compound in a blood
sample, comprising:
providing a measuring cell having at least first and second
electrodes for contact with the blood sample introduced into the
cell,
applying a potential to the electrodes to detect the presence of
the blood sample in the cell,
placing the blood sample into the cell,
removing the potential to the electrodes after the blood sample is
detected in the cell,
selectively oxidizing the compound in the blood 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 after the
selective oxidation of the compound in the blood sample has
substantially completed and measuring the resulting Cottrell
current, said current being proportional to the concentration of
the reduced electron acceptor and the selected compound in the
blood sample..Iaddend..Iadd.44. The method of claim 43, 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..Iaddend..Iadd.45. The method of claim 44, wherein the
Cottrell current is measured at the preset time after
re-application of a potential across the measuring cell
electrodes..Iaddend..Iadd.46. The method of claim 47, 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..Iaddend..Iadd.47. A method for measuring the amount
of glucose in blood, comprising:
providing a measuring cell with at least first and second
electrodes for contact with blood introduced into the cell,
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 after
the oxidation of glucose has substantially completed, and
measuring the Cottrell current through the cell, the Cottrell
current being proportional to the glucose concentration in the
blood..Iaddend..Iadd.48. The method of claim 46, wherein the
Cottrell current is measured at a preset time after re-application
of a potential across the measuring cell electrodes..Iaddend.
Description
FIELD OF THE INVENTION
The present invention relates to a disposable electroanalytical
cell and a method and apparatus for quantitatively determining the
presence of biologically important compounds such as glucose; TSH;
T4; hormones such as HCG; cardiac glycosides such as Digoxin;
antiarrhythmics such as Lidocaine; antiepileptics such as
phenobarbital; antibiotics such as Gentamicin; cholesterol;
non-therapeutic drugs and the like from body fluids.
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
two biologically important compounds--glucose and cholesterol.
WITH RESPECT TO GLUCOSE
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 injections of insulin,
and strict control of dietary intake, the prognosis for diabetics
is excellent. However, the blood glucose levels must 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.
At the 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 colorimetric methods which utilize the natural
oxidant of glucose to gluconic acid, specifically oxygen, are based
upon the reactions:
WITH RESPECT TO CHOLESTEROL
Current technology for the determination of cholesterol is also
based upon similar methods. In the case of cholesterol, the methods
presently used are based upon the generalized reactions:
In all present techniques, Dioxygen is the only direct oxidant used
with the enzyme cholesterol oxidase for the determination of both
free and total cholesterol. Using conventional test methods, oxygen
must diffuse into the sensor solution during use from the
surrounding air in order to provide sufficient reagent for a
complete reaction with the analyte cholesterol in undiluted serum
and whole blood specimens.
In both instances, the presence of the substance is determined by
quantifying, either colorometrically or otherwise, the presence of
hydrogen peroxide. The present methods of detection may include
direct measurement of the hydrogen peroxide produced by either
spectroscopic or electrochemical means and indirect methods in
which the hydrogen peroxide is reacted with various dyes, in the
presence of the enzyme peroxidase, to produce a color that is
monitored.
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.
These steps are dependent upon good and consistent operating
technique requiring strict attention to timing. Moreover, even
utilizing good operating technique, colorimetric 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 colorimetric
measurement vary widely in their calibration methods: some provide
no user calibration while others provide secondary standards.
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.
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.
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 amperometry 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.
The present invention address these requirements by providing
miniaturized disposable electroanalytic sample cells for precise
micro-aliquote 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.
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.
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 electro-chemical reduction
step suitable for quantifying the reaction production of the first
step. One advantage to utilizing an oxidant other than dioxygen for
the direct determination of an analyte is that they 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).
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.
In a presently preferred embodiment, oxidations with one electron
acceptor using ferricyanide, ferricinium, cobalt (III)
.[.orthophenantroline.]. .Iadd.orthophenanthroline.Iaddend., and
cobalt (III) dipyridyl are preferred. Benzoquinone is a two
electron acceptor which also provides excellent electro-oxidation
characteristics for amperometric quantitation.
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.
In this method of quantification, the measurement of a diffusion
controlled current at an accurately specified time (e.g. 20, 30, or
50 seconds, for example) after the instant of application of a
potential has the applicable equation for amperometry at a
controlled potential (E=constant) of: ##EQU1## where i denotes
current, nF is the number of coulombs per mole, A.[.D.]. is the
area of the electrode.Iadd., .Iaddend.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.
The method and apparatus of the .[.prevent.]. .Iadd.present
.Iaddend.invention permit, in preferred embodiments, direct
measurements of blood glucose, cholesterol and the like.
Furthermore, the sample cell according to the .[.prevent.].
.Iadd.present .Iaddend.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.
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.
The basic chemical binary reaction utilized by the method according
to the present invention is:
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 acceptors such as ferricyanide (wherein
the redox couple would be Fe(CN).sub.6.sup.-3
/Fe(CN).sub.6.sup.-4), ferricinium, cobalt III
.[.orthophenantroline.]. .Iadd.orthophenanthroline .Iaddend.and
cobalt (III) dipyridyl.
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 a 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.
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 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.
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.
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
another for measuring cholesterol which is to be taken in
conjunction with the accompanying drawings in which like numerals
indicate like components and in which:
FIG. 1 is an exploded view of a portable testing apparatus
according to the present invention;
FIG. 2 is a plan view of the sampling cell of the present
invention;
FIG. 3 is an exploded view of the sample cell shown in FIG. 2;
FIG. 4 is an exploded view of another embodiment of a sample cell
according to the invention;
FIG. 5 is a plan view of the cell shown in FIG. 4;
FIG. 6 is still another embodiment of a sample cell;
FIG. 7 is a graph showing current as a function of glucose
concentration;
FIG. 8 is a graphical presentation of Cottrell current as a
function of glucose concentration; and
FIG. 9 is a presently preferred circuit diagram of an electrical
circuit for use in the apparatus shown in FIG. 1.
FIG. 10 is a preferred embodiment of the electrochemical cell.
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 being operation when a sample cell 20 (FIG. 2) is
inserted into the window 19 of the apparatus.
With reference to FIGS. 2 and 3.Iadd., .Iaddend.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. Positioned on second substrate 23 is
reference electrode 24. Reference electrode 24 may be preferably
manufactured, for example, by vapor depositing the electrode onto a
substrate made from a material such as the polyimide Kapton. In the
preferred embodiment, reference electrode 24 is a silver-silver
chloride electrode. This electrode can be produced by .[.first.].
depositing a silver .Iadd.chloride .Iaddend.layer on a silver
.[.chloride.]. 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:
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:
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.
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 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.
Indicator or working electrode 26 can be either a strip of
platinum, gold, or palladium metallized plastic positioned on
reference electrode 24 or alternately the working electrode 26 and
the reference electrode may be manufactured as a coplanar unit with
electrode 26 being sandwiched between coplanar electrode 24
material. Preferable, sample cell 20 is prepared by sandwiching or
laminating the electrodes between the substrate to form a composite
unit.
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.
Referring to FIGS. 4-6, 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.
In FIG. 6, working electrode 226 is folded, thereby providing
increased surface area around opening 221, to achieve increased
sensitivity or specificity. In this case, reference electrode 224
is positioned beneath working electrode 226. Working electrode
includes cut out 234 to permit electrical contact with reference
electrode 224 through cut out 231 in substrate 222. End 230 of
substrate 222 also includes cut out 232 to permit electrical
contact with working electrode 226.
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.
.[.The.]. .Iadd.To .Iaddend.fully take advantage of the above
apparatus, the needed chemistry for the sell 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. When a supporting matrix is
used, it 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 .mu.m 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 adsorbed 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.
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 non-woven fabric or adsorbant 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.
For glucose determination, cells utilizing the coplanar design were
constructed having the reagent layer containing the following
formulations:
______________________________________ Glucose oxidase 600 u/ml
Potassium Ferricyanide 0.4M Phosphate Buffer 0.1M Potassium
Chloride 0.5M Gelatin 2.0 g/dl
______________________________________
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 place 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.
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.
Determination 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:
where t denotes time.
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. 8.
Proportionality between glucose concentrations and Cottrell
currents (recorded at t=30 seconds after the application of
potential) is shown in FIG. 7.
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.
The results of these many tests demonstrates the
microchronoamperometric methodology of the present invention and
its practicality for glucose self-monitoring by diabetics.
In a presently preferred embodiment of the invention utilizing
ferrocyanide, a number of tests were run showing certain improved
operating capabilities.
Referring to FIG. 9, 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 switch 43. Current from the electrodes W and R is converted
to a voltage by op amp 45. That voltage is converted into a digital
signal (frequency) by and 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 obviously be utilized
to obtain the advantages of the present invention.
With regard to FIG. 10, cell 400 consists of coplanar working 426
and reference 424 electrodes laminated between an upper 422 and
lower 423 nonconducting 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. At one end of cell 400 is an open area 427 similar to end
position 27 of FIG. 2.
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.
______________________________________ GLUCOSE SYSTEM COMPARISONS
Present Inven- Steps 1 2 3 4 tion
______________________________________ 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)
10-400 40-400 25-450 40-400 0-1000 CV** Hypoglycemic 15% 15% 5%
Euglycemic 10% 10% 3% Hyperglycemic 5% 5% 2% Correlation 0.921
0.862 0.95 ______________________________________ *RS--Reflectance
Spectroscopy **Coefficient of variation
With specific regard to the determination of cholesterol utilizing
the present invention, the generalized chemistry may be depicted
as: ##STR1## where the enzymes cholesterol esterase (CE) and
cholesterol oxidase (CO) catalyze reactions 1 and 2 respectively
and CO permits electron transfer with a variety of electroactive
couples (Ox and Red). Reaction 2 is novel in that electron
acceptors other than dioxygen may be used to oxidize cholesterol in
the presence of the enzyme cholesterol oxidase. Reaction 1 is well
known to those in the field and is necessary for the determination
of total cholesterol (free cholesterol and cholesterol esters).
Reaction 3 is an electro-oxidation process for probing and
quantitating the cholesterol.
Utilizing alternative oxidants according to the present invention,
the specific reactions become: ##STR2##
Cholesterol oxidase (CO) from a variety of sources will catalyze
electron transfer from cholesterol to a variety of the oxidants
including benzoquinone, benzoquinone derivatives such as
methylbenzoquinone, ethylbenzoquinone, chlorobenzoquinone,
ortho-benzoquinone (oxidized form of catechol),
benzoquinonesulfonate, and potassium ferricyanide. It is also
anticipated that the enzyme will allow electron transfer with other
alternate oxidants. As indicated in Reaction 3, the reduced product
can then be monitored amperometrically for the quantitative
determination of cholesterol.
Sources of the enzyme catalyzing the oxidation of cholesterol with
alternate oxidants include CO from Nocardia, Streptomyces,
Schizophyllum, Pseudomonas, and Brevibacterium; experimental
conditions under which it is able to rapidly catalyze the oxidation
of cholesterol by benzoquinone or any of the other oxidants depend
somewhat upon the source of the enzyme. For example, CO from
Streptomyces rapidly catalyzes substrate oxidation with
benzoquinone in phosphate buffer in the presence of any of a
variety of the surfactants including octylgluconopyranoside and
CHAPSO; the same reaction under identical conditions with CO from
either Brevibacterium or Nocardia is slower. However, both Nocardia
and Brevibacterium sources are active catalysts for cholesterol
oxidation by alternate oxidants under other conditions.
The oxidant also plays a role in which the enzyme is most active.
For example, cholesterol oxidase from Nocardia rapidly catalyzes
substrate oxidation with benzoquinone in 0.2 molar TRIS buffer and
3 g/dL CHAPSO but is slower with ferricyanide under identical
conditions; the Brevibacterium source of the enzyme is relatively
inactive with ferricyanide in TRIS buffer with a variety of
surfactants but when benzoquinone is used as the oxidant the
reaction is very fast. Alternatively, the Schizophyllum source of
the enzyme CO rapidly catalyzes the oxidation of cholesterol in
phosphate buffer with either ferricyanide or benzoquinone and with
a variety of surfactants as activators.
As indicated, cholesterol oxidase will catalyze the oxidation of
cholesterol by ferricyanide. Additional examples where CO catalyzes
cholesterol oxidation by ferricyanide include a Nocardia source in
TRIS buffer with a variety of surfactants including sodium
deoxycholate, sodium taurodeoxycholate, CHAPS, Thesit, and CHAPSO.
Furthermore, CO from Nocardia will also catalyze substrate
oxidation with ferricyanide in phosphate buffer with sodium
dioctylsulfosuccinate, sodium deoxycholate, sodium
taurodeoxycholate, and Triton X-100. The buffer concentration is
from 0.1 to 0.4 molar. Surfactant concentration for maximum
activity of the oxidase enzyme varies with each detergent. For
example, with deoxycholate or taurodeoxycholate, the enzyme in 0.2M
TRIS is most active with detergent in the range from 20 to 90
millimolar. However, enzyme catalytic activity is observed up to
.[.an.]. .Iadd.and .Iaddend.through a 10% concentration. With
octyl-gluconopyranoside, the maximum activity of the enzyme with
the oxidant ferricyanide occurs at a detergent concentration of
approximately 1.2%; however, the enzyme still maintains activity at
higher and lower concentrations of the surfactant.
Both esterase and CO require a surfactant for high activity.
Specific surfactants include sodium deoxycholate, sodium
taurodeoxycholate, sodium glycodeoxycholate, CHAPS
(3-(3-chlolamidopropyl)dimethylammonio-1-propanesulfonate), CHAPSO
(3-(3-chlolamidopropyl)dimethylammonio-2-hydroxy-1-propanesulfonate),
octyl-gluconopyranoside, octylthio-gluconopyranoside,
nonyl-gluconopyranoside, dodecyl-gluconopyranoside, Triton X-100,
Dioctyl sulfosuccinate, Thesit (Hydroxypolyethoxydodecane), and
lecithin (phosphatidylcholine). Buffers acceptable for this
reaction to occur with the enzyme include phosphate, TRIS, MOPS,
MES, HEPES, Tricine, Bicine, ACES, CAPS, and TAPS. An alternate
generallized reaction scheme for the measurement of cholesterol in
serum and other biological fluids is given ##STR3## where Ox, and
Red.sub.2 function as an electron mediator couple between the
cholesterol and the electroactive couple Ox.sub.2 /Red.sub.2. In
this case Ox.sub.1 and Red.sub.1 need not be electroactive because
they do not have to participate in the electrooxidation process
(Reaction 6). However, from both a thermodynamic and kinetic
perspective, this couple with the assistance of the enzyme
cholesterol oxidase must be able to accept electrons from
cholesterol and relay them to the electroactive couple (Ox.sub.2
/Red.sub.2). Specific examples of this chemistry include
EXAMPLE 1 ##STR4## Scheme II is beneficial when the rate of
reaction of cholesterol with the electroactive oxidant as in Scheme
I is so slow that it precludes its use in a practical sensor. As
mentioned above, Scheme II is also beneficial when the electron
mediator itself (Ox.sub.1 /Red.sub.1) is either not electroactive
or exhibits poor electrochemistry under conditions of the enzyme
chemistry. It is under these conditions that Scheme II is
particularly applicable. Other electron mediators (Ox.sub.1
/Red.sub.1) between cholesterol and ferricyanide for use in Scheme
II may be possible including phenazine ethosulfate, phenazine
methosulfate, tetramethylbenzidine, derivatives of benzoquinone,
naphthoquinone and naphthoquinone derivatives, anthraquinone and
anthraquinone derivatives, catechol, phenylenediamine,
.[.tetramethylphenenediamine.].
.Iadd.tetramethylphenylenediamine.Iaddend., and other derivatives
of phenylenediamine.
Furthermore, while it is understood that the oxidized form of the
electron relay accepts electrons from cholesterol, in the sensor
either the oxidized or the reduced form of the mediator may be
incorporated provided it reacts rapidly with both cholesterol and
ferricyanide. If the reduced form is sufficiently stable and the
oxidized form is not, then reductant, may be incorporated into the
sensor in relatively small quantity (in comparison with the analyte
to be determined) and still provide the electron relay. However,
this causes a corresponding background signal that must be
accounted for. The reductant, must also be isolated from
ferricyanide in the sensor by incorporation into a separate reagent
layer.
Several formulations of the above chemistries encompassing both
Schemes I and II have been prepare as dry films on membranes. These
membranes are positioned in the sensor which can then be used for
the determination of cholesterol. A preferred formulation of the
reagents involving Scheme II consists of the following
Cholesterol Esterase @400 Units/mL
Cholesterol Oxidase from Streptomyces @200 Units/mL
0.05 molar Potassium Ferricyanide
0.5 molar Potassium Chloride
0.2 molar Phosphate, pH 6.9
3 g/dL CHAPSO
2 g/dL gelatin
and 0.0001 molar hydroquinone (in the spreading or wicking
layer).
The concentractions provided are .[.that.]. .Iadd.those .Iaddend.of
the solutions which are coated onto porous supports, filter paper
or membranes; .[.these.]. .Iadd.those .Iaddend.concentrations are
reestablished when the membrane imbibes the serum or whole blood
specimen. For cholesterol determinations large pore sizes in the
filter support are .Iadd.more .Iaddend.necessary than that used for
glucose. This is because the cholesterol resides in the serum in
large lipoproteins (chylomicrons.[...]. .Iadd., .Iaddend.LDL, VLDL,
and HDL) which must penetrate the various layers of the sensor
until they reach the reagents. The surfactants to the major extent
break these natural micelles up into smaller micelles providing a
greater total surface .[.are.]. .Iadd.area .Iaddend.on which the
enzymes catalyze the reaction. Due to the instability of
benzoquinone.Iadd., .Iaddend.a small quantity of hydroquinone,
which is more stable by nature of its lower vapor pressure, is
incorporated into the sensor to assist electron mediation between
cholesterol and ferricyanide. Upon introduction of the serum
specimen into the sensor.Iadd., .Iaddend.the hydroquinone is
oxidized to benzoquinone; the benzoquinone is then free to pick up
electrons from the substrate and cycle them to ferricyanide. Under
these conditions the rate of the reaction of cholesterol with a
small quantity of benzoquinone is more rapid than that with a large
excess of ferricyanide.
An alternate and preferred formulation of reagents utilizing Scheme
II that may be incorporated into the reagent layer of the sensor
is:
Cholesterol Oxidase from Streptomyces @200 Units/mL
Lipase from Candida @500 Units/mL
3 g/dL CHAPSO
0.2 molar TRIS, pH 7.5
0.05 molar Potassium Ferricyanide
0.5 molar Potassium Chloride
0.05 molar MgCl.sub.2
2 g/dL gelatin
and 0.001 molar hydroquinone (in the spreading layer).
The magnesium salt in this formulation increases stability of the
esterase enzyme in the phosphate-free reagent layer; Lipase assists
the break up of the lipoproteins. With these dry reagent layers
incorporated into the sensor and using the evaluation methodology
as described, the following results were obtained.
______________________________________ Serum Cholesterol, mg %
Average Current, uA ______________________________________ 91 19.3
182 27.2 309 38.5 ______________________________________
These results demonstrate the quantitative response of the sensor
to serum cholesterol levels.
Alternate and preferred embodiment of the sensor utilizing Scheme I
is provided by reagent compositions:
Cholesterol Esterase @400 Units/mL
Cholesterol Oxidase from Nocardia @200 Units/mL
1 g/dL Triton X-100
0.1 molar TRIS buffer, pH 8.6
0.2 molar Potassium Ferricyanide
0.5 molar Potassium Chloride
0.02 molar MgCl.sub.2
2 g/dL gelatin
OR
Cholesterol Esterase @200 Units/mL
Cholesterol Oxidase from Streptomyces @200 Units/mL
0.06 molar Sodium deoxycholate
0.1 molar TRIS buffer, pH 8.6
0.2 molar Potassium Ferricyanide
0.5 molar Potassium Chloride
2 g/dL gelatin.
Thus, while we have illustrated and described the preferred
embodiment of my 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 invention of 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
defined and limited only by the claims which follow.
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.
* * * * *