U.S. patent application number 12/316142 was filed with the patent office on 2009-06-11 for reagents and methods for detecting analytes.
This patent application is currently assigned to Bayer HealthCare LLC. Invention is credited to Amy H. Chu, Karen L. Marfurt, Brenda L. Tudor, Mary Ellen Warchal-Windham, Boru Zhu.
Application Number | 20090145775 12/316142 |
Document ID | / |
Family ID | 40329165 |
Filed Date | 2009-06-11 |
United States Patent
Application |
20090145775 |
Kind Code |
A1 |
Chu; Amy H. ; et
al. |
June 11, 2009 |
Reagents and methods for detecting analytes
Abstract
A reagent for detecting an analyte comprises a flavoprotein
enzyme, a mediator such as a phenothiazine mediator, at least one
surfactant, a polymer and a buffer. The reagent may be used with an
electrochemical test sensor that includes a plurality of
electrodes.
Inventors: |
Chu; Amy H.; (Elkhard,
IN) ; Marfurt; Karen L.; (Edwardsburg, MI) ;
Tudor; Brenda L.; (Elkhart, IN) ; Warchal-Windham;
Mary Ellen; (Osceola, IN) ; Zhu; Boru;
(Granger, IN) |
Correspondence
Address: |
NIXON PEABODY LLP
161 N. CLARK STREET, 48TH FLOOR
CHICAGO
IL
60601
US
|
Assignee: |
Bayer HealthCare LLC
|
Family ID: |
40329165 |
Appl. No.: |
12/316142 |
Filed: |
December 10, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61007126 |
Dec 10, 2007 |
|
|
|
Current U.S.
Class: |
205/777.5 ;
204/403.14; 435/26 |
Current CPC
Class: |
C12Q 1/005 20130101;
G01N 27/3271 20130101; C12Q 1/004 20130101 |
Class at
Publication: |
205/777.5 ;
204/403.14; 435/26 |
International
Class: |
C12Q 1/32 20060101
C12Q001/32; G01N 27/26 20060101 G01N027/26 |
Claims
1. A reagent for detecting an analyte, the reagent comprising: a
flavoprotein enzyme; a mediator selected from the group
##STR00007## or a combination thereof, wherein R.sup.1, R.sup.2,
R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8, and R.sup.9
are the same or different, and are independently selected from the
group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl,
heteroaryl, cyclic, heterocyclic, halo, haloalkyl, carboxy,
carboxyalkyl, alkoxycarbonyl, aryloxycarbonyl, aromatic keto,
aliphatic keto, alkoxy, aryloxy, nitro, dialkylamino, aminoalkyl,
sulfo, dihydroxyboron, and combinations thereof; at least one
surfactant; a polymer; and a buffer, wherein at least one of the
surfactant and the buffer includes an inorganic salt, the ratio of
the total inorganic salt to mediator is less than about 3:1.
2. The reagent of claim 1, wherein the flavoprotein enzyme is
FAD-glucose dehydrogenase.
3. The reagent of claim 1, wherein the mediator includes
3-(2',5'-disulfophenylimino)-3H-phenothiazine.
4. The reagent of claim 1, wherein the surfactant includes a
saccharide-based surfactant or a phosphorylcholine-based
surfactant.
5. The reagent of claim 1, wherein the polymer is a cellulose-based
polymer.
6. The reagent of claim 1, wherein the buffer includes a phosphate
buffer.
7. The reagent of claim 1, wherein the ratio of the total inorganic
salt to mediator is in a ratio of less than about 2:1.
8. A reagent for detecting an analyte in a fluid sample, the
reagent comprising: FAD-glucose dehydrogenase having an activity of
from about 0.1 Units/.mu.L to about 10 Units/.mu.L; a
3-(2',5'-disulfophenylimino)-3H-phenothiazine mediator having a
concentration of from about 5 mM to about 120 mM; a surfactant
having a concentration of from about 0.05 wt. % to about 0.5 wt. %
of the reagent; a hydroxyethyl cellulose polymer having a
concentration of from about 0.1 wt. % to about 4 wt. % of the
reagent; and a buffer, wherein at least one of the surfactant and
the buffer includes an inorganic salt, the ratio of the total
inorganic salt to mediator is less than about 2:1.
9. The reagent of claim 8, further comprising a phosphate
buffer.
10. The reagent of claim 8, wherein the surfactant is a
heptanoyl-N-methylglucamide surfactant.
11. The reagent of claim 8, wherein the reagent comprises
FAD-glucose dehydrogenase having an activity of from about 0.5
Units/.mu.L to about 2.5 Units/.mu.L, a
3-(2',5'-disulfophenylimino)-3H-phenothiazine mediator having a
concentration of from about 30 mM to about 60 mM, a
heptanoyl-N-methylglucamide surfactant having a concentration of
from about 0.1 wt. % to about 0.4 wt. % of the reagent, and a
hydroxyethyl cellulose polymer having a concentration of from about
0.2 wt. % to about 0.5 wt. % of the reagent.
12. An electrochemical test sensor comprising: a working electrode
having a surface; a counter electrode having a surface; and a
reagent coating at least a portion of the surface of the working
electrode and at least a portion of the surface of the counter
electrode, the reagent comprising a flavoprotein, a phenothiazine
or a phenoxazine mediator, a buffer, and at least one surfactant, a
polymer and a buffer, wherein at least one of the surfactant and
the buffer includes an inorganic salt, the ratio of the total
inorganic salt to mediator is less than about 3:1.
13. The sensor of claim 12, wherein the flavoprotein includes
FAD-glucose dehydrogenase.
14. The sensor of claim 12, wherein the phenothiazine mediator
includes 3-(2',5'-disulfophenylimino)-3H-phenothiazine.
15. The sensor of claim 12, wherein the at least one surfactant
includes a heptanoyl-N-methylglucamide.
16. The sensor of claim 12, wherein the ratio of the total
inorganic salt to mediator is in a ratio of less than about
2:1.
17. A method of detecting an analyte in a fluid sample, the analyte
undergoing a chemical reaction, the method comprising the acts of:
providing an electrode surface; facilitating flow of the fluid
sample to the electrode surface using at least one surfactant;
catalyzing the chemical reaction with a flavoprotein enzyme;
generating a redox equivalent by the chemical reaction; and
transferring the redox equivalent to the electrode surface using a
phenothiazine or a phenoxazine mediator, wherein the maximum
kinetic performance is less than about 3 seconds.
18. The method of claim 17, wherein the electrode surface includes
a working electrode and a counter electrode, the electrode surface
including a reagent comprising the at least one surfactant, the
flavoprotein enzyme, the phenothiazine mediator, and a buffer.
19. The method of claim 18, wherein the reagent further includes a
polymer.
20. The method of claim 17, wherein the buffer includes a phosphate
buffer.
21. The method of claim 17, wherein the maximum kinetic performance
is less than about 2 seconds.
22. A method of detecting an analyte in a fluid sample, the method
comprising the acts of: providing an electrode surface; providing a
reagent including a flavoprotein enzyme; a mediator selected from
the group ##STR00008## or a combination thereof wherein R.sup.1,
R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8, and
R.sup.9 are the same or different, and are independently selected
from the group consisting of hydrogen, alkyl, alkenyl, alkynyl,
aryl, heteroaryl, cyclic, heterocyclic, halo, haloalkyl, carboxy,
carboxyalkyl, alkoxycarbonyl, aryloxycarbonyl, aromatic keto,
aliphatic keto, alkoxy, aryloxy, nitro, dialkylamino, aminoalkyl,
sulfo, dihydroxyboron, and combinations thereof; at least one
surfactant; and a buffer; the reagent being in contact with the
electrode surface; contacting the fluid sample with the reagent;
and determining the concentration of the analyte, wherein the
maximum kinetic performance is less than about 3 seconds.
23. The method of claim 22 wherein the reagent further includes a
polymer.
24. The method of claim 22 wherein the reagent includes a
phenothiazine mediator.
25. The method of claim 22 wherein at least one of the surfactant
and the buffer includes an inorganic salt, the ratio of the total
inorganic salt to mediator is less than about 3:1.
26. The method of claim 25 wherein at least one of the surfactant
and the buffer includes an inorganic salt, the ratio of the total
inorganic salt to mediator is less than about 2:1.
27. The method of claim 22 wherein the maximum kinetic performance
is less than about 2 seconds.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application Nos. 61/007,126, filed Dec. 10, 2007, the contents of
which are incorporated entirely herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates generally to reagents,
methods, and devices for measurement of analytes. More
particularly, the present invention relates to reagents, methods,
and devices for the measurement of glucose in a blood sample.
BACKGROUND OF THE INVENTION
[0003] The quantitative determination of analytes in body fluids is
of great importance in the diagnoses and maintenance of certain
physiological physical conditions. For example, lactate,
cholesterol, and bilirubin should be monitored in certain
individuals. In particular, it is important that individuals who
have diabetes frequently check the glucose level in their body
fluids to regulate the glucose intake in their diets. The results
of such tests may be used to determine what, if any, insulin or
other medication should be administered. In one type of
blood-glucose testing system, test sensors are used to test a
sample of blood.
[0004] A test sensor contains biosensing or reagent material that
reacts with, for example, blood glucose. The testing end of the
sensor is adapted to be placed into the fluid being tested (e.g.,
blood that has accumulated on a person's finger after the finger
has been pricked). The fluid may be drawn into a capillary channel
that extends in the sensor from the testing end to the reagent
material by capillary action so that a sufficient amount of fluid
to be tested is drawn into the sensor. The tests are typically
performed using optical or electrochemical testing methods.
[0005] Electrochemical test sensors are based on enzyme-catalyzed
chemical reactions involving the analyte of interest. In the case
of glucose monitoring, the relevant chemical reaction is the
oxidation of glucose to gluconolactone or its corresponding acid.
This oxidation is catalyzed by a variety of enzymes; some of which
may use coenzymes such as nicotinamide adenine dinucleotide
(phosphate) (NAD(P)), while others may use coenzymes such as flavin
adenine dinucleotide (FAD) or pyrroloquinolinequinone (PQQ).
[0006] In test sensor applications, the redox equivalents generated
in the course of the oxidation of glucose are transported to the
surface of an electrode, whereby an electrical signal is generated.
The magnitude of the electrical signal is then correlated with
glucose concentration. The transfer of redox equivalents from the
site of chemical reaction in the enzyme to the surface of the
electrode is accomplished using electron transfer mediators.
[0007] Electron transfer mediators previously used with FAD-glucose
dehydrogenase (FAD-GDH) include potassium ferricyanide,
phenazine-methosulfate (PMS), methoxy phenazine-methosulfate,
phenazine methyl sulfate, and dichloroindophenol (DCIP). These
compounds, however, have proven to be highly susceptible to the
environmental conditions including temperature and moisture, which
result in test sensor reagents of low stability. For example,
during storage, reduced mediator may be produced from interactions
between the oxidized mediator and the enzyme system. The larger the
amount of mediator or enzyme, the larger the amount of reduced
mediator that is produced. The background current, which increases
over time, will generally increase toward the end of the shelf-life
of the sensor strips because of the high concentration of reduced
mediator. The increased background current may decrease the
precision and accuracy of the measurements of the test sensor and,
thus, provide a limited shelf-life for the test sensors.
[0008] Another disadvantage associated with existing test sensors
is the relatively slow fill rate. Achieving a fast sensor fill rate
is desirable so that the re-hydration of the reagent may be faster
and more uniform. Thus, faster fill rates generally result in more
precise, stable test sensors having less variation.
[0009] Therefore, it would be desirable to have a reagent that
addresses one or more of these disadvantages.
SUMMARY OF THE INVENTION
[0010] According to one aspect of the present invention, a reagent
for detecting an analyte comprises a flavoprotein enzyme, a
mediator selected from the group
##STR00001##
[0011] or a combination thereof. R.sup.1, R.sup.2, R.sup.3,
R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8, and R.sup.9 are the
same or different and are independently selected from the group
consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl,
cyclic, heterocyclic, halo, haloalkyl, carboxy, carboxyalkyl,
alkoxycarbonyl, aryloxycarbonyl, aromatic keto, aliphatic keto,
alkoxy, aryloxy, nitro, dialkylamino, aminoalkyl, sulfo,
dihydroxyboron, and combinations thereof. The reagent further
comprises at least one surfactant, a polymer and a buffer. At least
one of the surfactant and the buffer includes an inorganic salt in
which the ratio of the total inorganic salt to mediator is less
than about 3:1.
[0012] According to another embodiment of the present invention, a
reagent for detecting an analyte in a fluid sample includes
FAD-glucose dehydrogenase having an activity of from about 0.1
Units/.mu.L to about 10 Units/.mu.L. The reagent further comprises
a 3-(2',5'-disulfophenylimino)-3H-phenothiazine mediator having a
concentration of from about 5 mM to about 120 mM. The reagent
further comprises a surfactant having a concentration of from about
0.05 wt. % to about 0.5 wt. % of the reagent. The reagent further
comprises a hydroxyethyl cellulose polymer having a concentration
of from about 0.1 wt. % to about 4 wt. % of the reagent and a
buffer. At least one of the surfactant and the buffer includes an
inorganic salt in which the ratio of the total inorganic salt to
mediator is less than about 3:1.
[0013] According to another embodiment of the present invention, an
electrochemical test sensor comprises a working electrode having a
surface. The test sensor further comprises a counter electrode
having a surface. The test sensor further comprises a reagent
coating at least a portion of the surface of the working electrode
and at least a portion of the surface of the counter electrode. The
reagent comprises a flavoprotein, a phenothiazine or a phenoxazine
mediator, a buffer, and at least one surfactant and a polymer. At
least one of the surfactant and the buffer includes an inorganic
salt in which the ratio of the total inorganic salt to mediator is
less than about 3:1.
[0014] According to one process of the present invention, a method
of detecting an analyte in a fluid sample, the analyte undergoing a
chemical reaction, comprises the act of providing an electrode
surface. The method further comprises the act of facilitating flow
of the fluid sample to the electrode surface using a surfactant.
The method further comprises the act of catalyzing the chemical
reaction with a flavoprotein enzyme. The method further comprising
the act of generating a redox equivalent by the chemical reaction.
The method further comprises the act of transferring the redox
equivalent to the electrode surface using a phenothiazine or a
phenoxazine mediator. The maximum kinetic performance is less than
about 3 seconds.
[0015] According to another method, an analyte is detected in a
fluid sample and includes providing an electrode surface. A reagent
is provided that includes a flavoprotein enzyme and a mediator is
selected from the group
##STR00002##
or a combination thereof, wherein R.sup.1, R.sup.2, R.sup.3,
R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8, and R.sup.9 are the
same or different, and are independently selected from the group
consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl,
cyclic, heterocyclic, halo, haloalkyl, carboxy, carboxyalkyl,
alkoxycarbonyl, aryloxycarbonyl, aromatic keto, aliphatic keto,
alkoxy, aryloxy, nitro, dialkylamino, aminoalkyl, sulfo,
dihydroxyboron, and combinations thereof; at least one surfactant;
and a buffer; the reagent contacting the electrode surface. The
fluid sample contacts the reagent. The concentration of the analyte
is determined. The maximum kinetic performance is less than about 3
seconds.
[0016] The above summary of the present invention is not intended
to represent each embodiment, or every aspect, of the present
invention. Additional features and benefits of the present
invention are apparent from the detailed description and figures
set forth below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1a is a test sensor according to one embodiment.
[0018] FIG. 1b is a side view of the test sensor of FIG. 1a.
[0019] FIG. 2 is a line graph plotting current measurements against
glucose concentrations.
[0020] FIG. 3 is a bar graph comparing fill times of sensors
including heptanoyl-N-methylglucamide (MEGA 8) surfactant with
sensors not including MEGA 8 surfactant.
[0021] FIG. 4 is a bar graph comparing background current of
sensors including MEGA 8 surfactant with sensors not including MEGA
8 surfactant.
[0022] FIG. 5 is a bar graph of fill times of test sensors versus
different formulations with and without surfactants.
[0023] FIG. 6 is a plot of measured current values versus time in a
formulation including a surfactant.
[0024] FIG. 7 is a bar graph of peak times using 50 mg/dL of
glucose, mediators with different sulfate concentrations and
different phosphate buffer concentrations.
[0025] FIG. 8 is a bar graph of peak times using 100 mg/dL of
glucose, mediators with different sulfate concentrations and
different phosphate buffer concentrations.
[0026] FIG. 9 is a bar graph of peak times using 400 mg/dL of
glucose, mediators with different sulfate concentrations and
different phosphate buffer concentrations.
[0027] FIGS. 10a, 10b are plots of measured current values versus
time in formulations having different inorganic salt
concentrations.
[0028] FIG. 11 is a graph with % CV for low salt reagent solutions
and high salt reagent formulations.
[0029] While the invention is susceptible to various modifications
and alternative forms, specific embodiments are shown by way of
example in the drawings and are described in detail herein. It
should be understood, however, that the invention is not intended
to be limited to the particular forms disclosed. Rather, the
invention is to cover all modifications, equivalents, and
alternatives falling within the spirit and scope of the
invention.
DESCRIPTION OF ILLUSTRATED EMBODIMENTS
[0030] The present invention is directed to reagents, methods, and
devices for measurement of analytes. More specifically, the present
invention is directed to a test sensor reagent for detecting an
analyte including (1) a flavoprotein enzyme, (2) a phenothiazine or
phenoxazine mediator, (3) a buffer, (4) a surfactant or a
combination of surfactants, and/or (5) a cellulose-based
polymer.
[0031] The reagents described herein may be used to assist in
determining an analyte concentration in a fluid sample. The nature
of the analyte monitored in accord with the present invention is
unrestricted, provided the analyte undergoes a chemical reaction
that is catalyzed by a flavoprotein enzyme. Some examples of the
types of analytes that may be collected and analyzed include
glucose, lipid profiles (e.g., cholesterol, triglycerides, LDL, and
HDL), microalbumin, hemoglobin, A.sub.1C, fructose, lactate, or
bilirubin. It is contemplated that other analyte concentrations may
be determined. The analytes may be in, for example, a whole blood
sample, a blood serum sample, a blood plasma sample, other body
fluids such as ISF (interstitial fluid), urine, and non-body
fluids.
[0032] The test sensors described herein are electrochemical test
sensors. Meters used with the electrochemical test sensors may have
optical aspects so as to detect the calibration information and
electrochemical aspects to determine information related to the
analyte (e.g., the analyte concentration) of the fluid sample. One
non-limiting example of an electrochemical test sensor is shown in
FIG. 1a. FIG. 1a depicts a test sensor 10 including a base 11, a
capillary channel, and a plurality of electrodes 16 and 18. A
region 12 shows an area that defines the capillary channel (e.g.,
after a lid is placed over the base 11). The plurality of
electrodes includes a counter electrode 16 and a working electrode
18. The electrochemical test sensor may also contain at least three
electrodes, such as a working electrode, a counter electrode, a
trigger electrode, or another electrode to detect interference
substances (e.g., hematocrit, ascorbate, uric acid) in the fluid
sample. The working electrode employed in electrochemical sensors
according to the embodiments of the present invention may vary,
with suitable electrodes including, but not limited to, carbon,
platinum, palladium, gold, combinations thereof, and the like.
[0033] The electrodes 16, 18 are coupled to a plurality of
conductive leads 15a,b, which, in the illustrated embodiment,
terminates with larger areas designated as test-sensor contacts
14a,b. The capillary channel is generally located in a
fluid-receiving area 19. Examples of electrochemical test sensors,
including their operation, may be found in, for example, U.S. Pat.
No. 6,531,040 assigned to Bayer Corporation. It is contemplated
that other electrochemical test sensors may be employed with the
embodiments of the present invention.
[0034] The fluid-receiving area 19 includes at least one reagent
for converting the analyte of interest (e.g., glucose) in the fluid
sample (e.g., blood) into a chemical species that is
electrochemically measurable, in terms of the electrical current it
produces, by the components of the electrode pattern. The reagent
typically includes an analyte-specific enzyme that reacts with the
analyte and with an electron acceptor to produce an
electrochemically measurable species that may be detected by the
electrodes. The reagent may include mediators or other substances
that assist in transferring electrons between the analyte and the
conductor, binders that hold the enzyme and mediator together,
other inert ingredients, or combinations thereof.
[0035] A fluid sample (e.g., blood) may be applied to the
fluid-receiving area 19. The fluid sample reacts with the at least
one reagent. After reacting with the reagent and in conjunction
with the plurality of electrodes, the fluid sample produces
electrical signals that assist in determining the analyte
concentration. The conductive leads 15a,b carry the electrical
signal back toward a second opposing end 42 of the test sensor 10
where the test-sensor contacts 14a,b transfer the electrical
signals into the meter.
[0036] Referring to FIG. 1b, a side view of the test sensor 10 of
FIG. 1a is shown. As shown in FIG. 1b, the test sensor 10 of FIG.
1b further includes a lid 20 and a spacer 22. The base 11, the lid
20, and the spacer 22 may be made from a variety of materials such
as polymeric materials. Non-limiting examples of polymeric
materials that may be used to form the base 11, the lid 20, and the
spacer 22 include polycarbonate, polyethylene terephthalate (PET),
polyethylene naphthalate (PEN), polyimide, and combinations
thereof. It is contemplated that other materials may be used in
forming the base 11, lid 20, and/or spacer 22.
[0037] To form the test sensor 10 of FIGS. 1a, 1b, the base 11, the
spacer 22, and the lid 20 are attached by, for example, an adhesive
or heat sealing. When the base 11, the lid 20, and the spacer 22
are attached, the fluid-receiving area 19 is formed. The
fluid-receiving area 19 provides a flow path for introducing the
fluid sample into the test sensor 10. The fluid-receiving area 19
is formed at a first end or testing end 40 of the test sensor 10.
Test sensors of the embodiments of the present invention may be
formed with a base and a lid in the absence of a spacer, where the
fluid-receiving area is formed directly in the base and/or the
lid.
[0038] Flavoproteins in accord with the present invention include
any enzymes having flavin cofactors. Some non-limiting examples of
flavoproteins include FAD-glucose oxidase (Enzyme Classification
No. 1.1.3.4), Flavin-hexose oxidase (EC No. 1.1.3.5) and
FAD-glucose dehydrogenase (EC No. 1.1.99.10). Additional oxidase
enzymes for use in accord with the present invention include, but
are not limited to, lactate oxidase, cholesterol oxidase, alcohol
oxidase (e.g., methanol oxidase), d-aminoacid oxidase, choline
oxidase, and FAD derivatives thereof A desirable flavoprotein for
use in accord with the present invention is FAD-glucose
dehydrogenase (FAD-GDH).
[0039] Mediators in accord with the present invention include
phenothiazines having the formula
##STR00003##
and phenoxazines having the formula
##STR00004##
wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6,
R.sup.7, R.sup.8, and R.sup.9 are the same or different and are
independently selected from the group consisting of hydrogen,
alkyl, alkenyl, alkynyl, aryl, heteroaryl, cyclic, heterocyclic,
halo, haloalkyl, carboxy, carboxyalkyl, alkoxycarbonyl,
aryloxycarbonyl, aromatic keto, aliphatic keto, alkoxy, aryloxy,
nitro, dialkylamino, aminoalkyl, sulfo, dihydroxyboron, and
combinations thereof. It is contemplated that isomers of the same
may also be formed.
[0040] One desirable example of a phenothiazine that has been
prepared and found to have suitable properties as an NADH mediator
is a water-soluble sodium or ammonium salt of 3-(2',5'
disulfophenylimino)-3H-phenothiazine having the formula
##STR00005##
3-(2',5'-disulfophenylimino)-3H-phenothiazine is associated with a
particularly low background current, which results in improved
signal-to-noise ratios. Another desirable example is
3-(3',5'-dicarboxy-phenylimino)-3H-phenothiazine mediator that has
been prepared and found to have suitable properties as an NADH
mediator. The background current of these phenothiazines was found
to be significantly less than previously-used mediators.
[0041] Other phenothiazines and phenoxazines that have been found
to have suitable properties as NADH mediators are
3-(4'-chloro-phenylimino)-3H-phenothiazine;
3-(4'-diethylamino-phenylimino)-3H-phenothiazine;
3-(4'-ethyl-phenylimino)-3H-phenothiazine;
3-(4'-trifluoromethyl-phenylimino)-3H-phenothiazine;
3-(4'-methoxycarbonyl-phenylimino)-3H-phenothiazine;
3-(4'-nitro-phenylimino-3H-phenothiazine;
3-(4'-methoxy-phenylimino)-3H-phenothiazine;
7-acetyl-3-(4'-methoxycarbonylphenylimino)-3H-phenothiazine;
7-trifluoromethyl-3-(4'-methoxycarbonyl-phenylimino)-3H-phenothiazine;
3-(4'-.omega.-carboxy-n-butyl-phenylimino)-3H-phenothiazine;
3-(4'-aminomethyl-phenylimino)-3H-phenothiazine;
3-(4'-(2''-(5''-(p-aminophenyl)-1,3,4-oxadiazoyl)phenylimino)-3H-phenothi-
azine; 3-(4'-p-aminoethyl-phenylimino)-3H-phenothiazine;
6-(4'-ethylphenyl)amino-3-(4'-ethylphenylimino)-3H-phenothiazine;
6-(4'-[2-(2-ethanoloxy)ethoxy]-ethoxyphenyl)amino-3-(4'-[2-(2-ethanoloxy)-
ethoxy]ethoxyphenylimino)-3H-phenothiazine;
3-(4'-[2-(2-ethanoloxy)ethoxy]ethoxy-phenylimino)-3H-phenothiazine;
3-(4'-phenylimino)-3H-phenothiazineboronic acid,
3-(3',5'-dicarboxy-phenylimino)-3H-phenothiazine;
3-(4'-carboxyphenylimino)-3H-phenothiazine;
3-(3',5-dicarboxy-phenylimino)-3H-phenoxazine;
3-(2',5'-phenylimino)-3H-phenothiazinedisulfonic acid; and
3-(3'-phenylimino)-3H-phenothiazinesulfonic acid.
[0042] In one embodiment, a
3-(2',5'-disulfophenylimino)-3H-phenothiazine mediator was prepared
by dissolving phenothiazine (1.53 mole, 1.1 equivalent, 306 g) with
stirring into 6.0 L of tetrahydrofuran (THF) and then cooled to
0.degree. C. Aniline 2,5-disulfonic acid (1.38 mole, 350 g) was
dissolved in 7.0 L of water and 1 M sodium hydroxide (NaOH) (128
ml) was added during stirring. The aniline 2,5-disulfonic acid
solution was added slowly, over the course of about 2 hrs, to the
phenothiazine solution, to give a white, cloudy suspension. The
phenothiazine/aniline suspension was at a temperature of about
0.degree. C. to about 4.degree. C. Sodium persulfate (5.52 mole, 4
equivalent, 1314 g) was dissolved in 4.0 L of water to form a
sodium persulfate solution.
[0043] The sodium persulfate solution was added dropwise over 3
hours to the phenothiazine/aniline suspension at a temperature
between about 0.degree. C. to about 3.degree. C. and resulted in a
very dark solution. The very dark solution remained cold using an
ice bath and was stirred overnight. The contents were then
transferred to a Buchi rotary evaporator and the tetrahydrofuran
was removed over the course of about 2 hours at a temperature less
than 35.degree. C. After the evaporation act, the remaining
solution was transferred to a 25 L separator and backwashed with
ethyl acetate. The remaining solution was backwashed 3 times using
2 L of ethyl acetate each time. The reaction fluids were cooled
while stirring to -3.degree. C. in an acetone/CO.sub.2 bath. The
precipitated solid was filtered through two cloths on two 24 cm
Buchner funnels on the same day. The precipitated solid was left
overnight in the funnels to dry and then transferred to a flask
containing 2 L of acetonitrile and stirred for about 1 hour at room
temperature. To remove the residual water, the sample was then
filtered and washed with more acetonitrile. The mediator was dried
to a constant weight in a vacuum oven at 35.degree. C.
[0044] Because of the low background current achieved using
reagents having 3-(2',5'-disulfophenylimino)-3H-phenothiazine
mediators, the same reagent formulation may be applied to both the
working electrode and the counter electrode of an electrochemical
test sensor. Applying the same reagent to both the working
electrode and the counter electrode simplifies the manufacturing
process and thereby decrease the costs associated therewith.
Additionally, the low background current assists in obtaining
accurate glucose readings, especially with samples having low
glucose concentrations, which is particularly important in
analyzing neonatal blood glucose assays.
[0045] The reagents of the embodiments of the present invention
further include a surfactant or a combination of surfactants,
and/or a cellulose-based polymer. The surfactant or combination of
surfactants facilitates the sensor blood fill rate and re-hydration
of a dry reagent. The faster blood fill rate and reagent
re-hydration rate are desirable for achieving a quicker assay
(e.g., less than 5-second assay) across an about 20% to an about
70% hematocrit range.
[0046] The surfactant is desirably selected from biocompatible ones
including saccharide-based surfactants or phosphorylcholine-based
surfactants. One non-limiting example of a saccharide-based
surfactant is heptanoyl-N-methylglucamide (MEGA 8 from
Sigma-Aldrich of St. Louis, Mo.). Surfactants such as MEGA 8 assist
in increasing the thermal stability of test sensors. Additionally,
surfactants such as MEGA 8 assist in fast fill rates, even for
blood samples having high hematocrit levels. Using surfactants such
as MEGA 8 with other inert ingredients, (e.g., hydroxyethyl
cellulose polymer and/or a neutral pH buffer) in a reagent
formulation provides sensors with great stability, even at elevated
temperatures. Non-limiting examples of phosphorylcholine-based
surfactants include the Lipidure series (NOF Corporation,
Japan).
[0047] Surfactants may also be selected from conventional neutral
surfactants such as ethoxylated oleyl alcohol (Rhodasurf ON870 from
Rhodia Inc. in Cranbury, N.J.). Surfactants may also be selected
from anionic surfactants such as sodium methyl cocoyl taurate
(Geropon TC-42 from Rhodia Inc.) and alkyl phenol ethoxylate
phosphate (Phospholan CS131 from Akzo-Nobel Surface Chemistry LLC
in Chicago, Ill.). It is contemplated that other surfactants may be
used in forming the reagent.
[0048] Alternatively or additionally, the reagents of the
embodiments of the present invention include a polymer. The
reagents may include a cellulose-based polymer such as hydroxyethyl
cellulose polymer. In some embodiments, the cellulose-based polymer
is a low to medium molecular weight cellulose-based polymer. The
polymer, such as a cellulose-based polymer, assists in providing
the reagent with increased stability and adequate viscosity so that
the reagent, when dried, stays in its original position on the
sensor substrate. It is contemplated that other polymers may be
used such as, for example, polyvinyl pyrrolidine (PVP).
[0049] The reagent may also include a buffer (e.g., a phosphate
buffer) and/or other inert components. Non-limiting examples of
suitable buffer solutions include but are not limited to Good's
buffers (e.g., HEPES (i.e.,
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), MOPS (i.e.,
3-(N-morpholino)propanesulfonic acid), TES (i.e.,
N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid)),
Mcllvaine's buffers, combinations thereof, or the like.
[0050] To provide a desirable assay precision, thermal stability
and maximum kinetic performance, the ratio of the inorganic salts
to mediator should be less than about 3:1. The source of inorganic
salts could be from the buffer and/or the mediator. It is even more
desirable for the ratio of the inorganic salts to mediator to be
less than about 2:1 or even less than about 1.5:1.
[0051] According to one embodiment of the present invention, a
reagent includes FAD-GDH, a low background phenothiazine mediator,
a surfactant or combination of surfactants, a cellulose-based
polymer, and a buffer to achieve improved sensor performance and
stability. The reagent may be used to determine the glucose
concentration in biological specimen such as blood, plasma, serum,
or urine. In one embodiment, the phenothiazine mediator is
3-(2',5'-disulfophenylimino)-3H-phenothiazine. In another
embodiment, the surfactant is MEGA 8 and the polymer is
hydroxyethyl cellulose. In one embodiment, a reagent includes
FAD-GDH having an activity ranging from about 0.1 Units/.mu.L to
about 10 Units/.mu.L, about 5 mM to about 120 mM of
3-(2',5'-disulfophenylimino)-3H-phenothiazine mediator, about 0.05
wt. % to about 0.5 wt. % of MEGA 8 surfactant, about 0.1 wt. % to
about 4 wt. % of hydroxyethyl cellulose, and about 25 mM to about
200 mM of buffer having a pH of about 4 to about 8. In another
embodiment, a reagent includes FAD-GDH having an activity ranging
from about 0.5 Units/.mu.L to about 2.5 Units/.mu.L, about 30 mM to
about 60 mM of 3-(2',5'-disulfophenylimino)-3H-phenothiazine
mediator, about 0.1 wt. % to about 0.4 wt. % of MEGA 8 surfactant,
0.01 to 0.1% of Geropon TC-42, about 0.2 wt. % to about 0.5 wt. %
of hydroxyethyl cellulose, and about 50 mM to about 150 mM of
buffer having a pH of about 6 to about 7.
Example 1
[0052] As shown in FIG. 2, the reactivity of the chemistry for four
sensor lots was analyzed by generating a glucose dose-response
curve for sensors including FAD-GDH enzyme having an activity of
about 1.75 Units/.mu.L, about 40 mM
3-(2',5'-disulfophenylimino)-3H-phenothiazine mediator, about 0.2
wt. % MEGA 8 surfactant, about 0.25 wt. % hydroxyethyl cellulose,
and about 100 mM phosphate buffer having a pH of about 6.5. The
sensors were tested with whole blood samples at about 40%
hematocrit level. The blood glucose concentrations of the blood
samples were about 0 mg/dL, 38 mg/dL, 67 mg/dL, 112 mg/dL, 222
mg/dL, 339 mg/dL, and 622 mg/dL. For each blood sample, ten
replicates were collected for each sensor lot. As shown in FIG. 2,
the mean current for each sample was plotted against the sample
glucose concentration (mg/dL) measured by a Yellow Springs Glucose
Analyzer (YSI, Inc., Yellow Springs, Ohio) for each sensor lot. The
slope of the dose response lines was about 20 nA/mg/dL, which
indicates relatively high sensitivity of the test sensors. The
y-intercepts were relatively close to 0 nA, which indicates low
background noise levels. These results indicate that accurate
readings may be achieved using the test sensors including the
reagent described herein.
[0053] The coefficient of variation was determined for each of the
ten replicates of the sensor lots used to generate the graph of
FIG. 2. Table 1 below shows the average coefficient of variation
percent (% CV) from the four sensor lots.
TABLE-US-00001 TABLE 1 Glucose Concentration 38 67 112 222 339 622
mg/dL mg/dL mg/dL mg/dL mg/dL mg/dL % CV 3.1 2.1 3.4 2.5 2.1
1.2
[0054] Because of the low background noise of the sensors including
the reagent of the embodiments of the present invention, the
average assay % CV was less than 3.5%, even for samples having low
glucose concentrations. Thus, the % CV values were well under 5%,
which is often considered to be the standard acceptable limit. This
low % CV indicates high precision of the test sensors.
Additionally, the low % CV is associated with low variance among
test sensors, which is desirable for obtaining consistent test
results.
Example 2
[0055] FIG. 3 shows a graph illustrating the affect of MEGA 8
surfactant on the sensor fill rate using 60% hematocrit whole
blood. The test sensors used in FIG. 3 included FAD-GDH having an
activity of about 1 Unit/.mu.L (about 192 Units/mg), about 4 wt. %
(about 120 mM) of potassium ferricyanide mediator, about 1.6 wt. %
of the reagent of 4 wt. % hydroxyethyl cellulose, and about 35 mM
of citrate buffer at a pH of about 5.0. Potassium ferricyanide
mediator was used to test whether MEGA 8 surfactant without
3-(2',5'-disulfophenylimino)-3H-phenothiazine mediator had
desirable affects on a test sensor. The fill rates of a group of
thirty test sensors including about 0.2 wt. % MEGA 8 surfactant
were compared against a control group of thirty test sensors not
including MEGA 8 surfactant. Initial fill rates of ten sensors from
each of the two groups of were measured. Ten sensors from each
group were then exposed to a temperature of about -20.degree. C.
for about two weeks. Finally, ten sensors from each group were
exposed to a temperature of about 50.degree. C. for about two
weeks. The average fill times of each group and subgroup of test
sensors were calculated and are shown in FIG. 3. The blood fills
the reaction chamber of the test sensor in less than 0.3 sec. with
the reagent having surfactant and a 60% hematocrit whole blood
sample. As shown in FIG. 3, the fill rate of the sensors including
MEGA 8 surfactant was at least twice as fast and up to about four
times faster than those sensors not including the MEGA 8
surfactant.
Example 3
[0056] The background currents of heat-stressed sensors including
MEGA 8 surfactant were compared to the background currents of
heat-stressed sensors not including MEGA 8 surfactant. The test
sensors used in FIG. 4 included FAD-GDH having an activity of about
1 Unit/.mu.L, about 50 mM of
3-(2',5'-disulfophenylimino)-3H-phenothiazine mediator, about 0.75
wt. % of the reagent of hydroxyethyl cellulose, and about 50 mM of
buffer at a pH of about 7. A first group of forty test sensors did
not include MEGA 8 surfactant. A second group of forty test sensors
included about 0.2 wt. % MEGA 8 surfactant. Each of the first and
second groups included two subgroups: a first subgroup including
twenty test sensors having FAD-GDH from Amano Enzyme Inc. (Nagoya,
Japan) and a second subgroup including twenty test sensors having
FAD-GDH from Toyobo Co. (Osaka, Japan) Ten of the test sensors from
each of the subgroups were stored at about 50.degree. C. for about
two weeks. The remaining test sensors were stored at about
-20.degree. C. for about two weeks. The background current of the
sensors was then tested using 40% hematocrit whole blood samples
having a glucose concentration of about 0 mg/dL. Ten replicates per
sample were collected. FIG. 4 shows a graph illustrating the mean
sensor background current from the ten replicates. As shown in FIG.
4, the test sensors including MEGA 8 surfactant had a significantly
lower sensor background current change as compared with the test
sensors not including MEGA 8 surfactant, indicating that the
reagent is more stable when MEGA 8 surfactant is added to the
reagent.
Example 4
[0057] The thermal stability of test sensors according to the
embodiments of the present invention were also tested. The test
sensors used in this example included FAD-GDH having an activity of
about 2 Units/.mu.L, about 40 mM of
3-(2',5'-disulfophenylimino)-3H-phenothiazine mediator, about 0.2
wt. % of MEGA 8 surfactant, about 0.25 wt. % of the reagent of
hydroxyethyl cellulose, and about 100 mM of buffer at a pH of about
6.5. A first group of test sensors was stored at about 50.degree.
C. for about two weeks. A second group of test sensors was stored
at about -20.degree. C. for about two weeks. The performance of the
sensors in each group was then evaluated with 40% hematocrit whole
blood samples having glucose concentrations of about 50 mg/dL,
about 100 mg/dL, and about 400 mg/dL. Ten replicates per sample
were collected. The mean difference in glucose concentration
between the test sensors stored at 50.degree. C. and those stored
at -20.degree. C. was calculated and compared to several different
types of self testing blood glucose monitoring systems. The glucose
assay bias of the test sensors according to the embodiments of the
present invention was negligible. Thus, there was no appreciable
change in the glucose assay results even after storing the sensors
at relatively extreme temperatures for two weeks. In contrast, the
glucose assay bias of the comparative commercially available test
sensors was generally from about 5% to about 12%. Thus, the thermal
stability of the test sensors of the embodiments of the present
invention was significantly better than that of existing test
sensors.
Example 5
[0058] Tests were performed using test sensors to determine fill
speed with high hematocrit blood samples. Specifically, as shown in
FIG. 5, a formulation using no surfactant (Formula 1) and
surfactants (Formulas 2-7) were tested. The formulations are listed
in Table 2 below.
TABLE-US-00002 TABLE 2 Formula 1 Formula 2 Formula 3 Formula 4
Formula 5 Formula 6 Formula 7 Mediator (mM) 60 60 60 60 60 50 90
Buffer (mM) 75 75 75 75 75 100 112 FAD-GDH (U/uL) 3.00 3.00 3.00
3.00 3.00 1.25 3.75 Polymer (HEC)(%) 0.38 0.38 0.38 0.38 0.38 0.63
0.60 MEGA 8 (wt. %) 0.10 0.05 0.225 Rhodasurf (wt. %) 0.10 0.05
Zwittergent 312 (wt. %) 0.30 Phospholan CS131 (wt. %) 0.10 Mediator
= 3-(2',5'-disulfophenylimino)-3H-phenothiazine Buffer = Phosphate
except Formula 6 used TES TES =
(N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid) HEC =
hydroxyethyl cellulose
Specifically, Formulas 2 and 3 included the surfactant MEGA 8,
while Formulas 4 and 5 included the surfactant Rhodasurf. Formula 6
included the surfactant Zwittergent and Formula 7 included
surfactants MEGA 8 and Phospholan CS 131. Formulas 1-7 were
stressed under two different conditions. Specifically, Formulas 1-7
were stressed for 2 weeks at a temperature of -20.degree. C. and
also stressed for 2 weeks at a temperature of 50.degree. C.
[0059] After being stressed at these conditions, Formulas 1-7 were
deposited onto electrodes on the test sensor. The sensors were
tested in a vertical (90.degree.) position with whole blood at
60-70% hematocrit. The sensors were videotaped during filling and
the time was measured. The times required for the high hematocrit
blood to fill the entire sensor reaction chamber for Formulas 1-7
are shown in FIG. 5. The sensor-fill time for Formula 1 (without
surfactant) was about 0.6 and 0.7 sec. for the sensors stored at
-20.degree. C. and 50.degree. C./2 wks., respectively. On the hand,
Formulas 2-7 had sensor-fill times for both -20.degree. C. and
50.degree. C./2 wks. were about 0.5 sec. or less. Most of the
Formulas 2-7 had sensor-fill times for both -20.degree. C. and
50.degree. C./2 wks. that were about 0.4 seconds or less with
several being less than about 0.3 sec. Thus, Formulas 2-7 with
surfactants had much improved fill times over Formula 1 without a
surfactant.
Example 6
[0060] A reagent was tested to determine its maximum kinetic
performance. The reagent included 40 mM of
3-(2',5'-disulfophenylimino)-3H-phenothiazine, 50 mM of phosphate
buffer, 2.00 U/ul of FAD-GDH, 0.25 wt. % of hydroxyethyl cellulose
(HEC) and 0.20 wt. % of the surfactant MEGA 8.
[0061] FIG. 6 shows the output signals from test sensors having
blood samples with a glucose concentration of 400 mg/dL and 70%
hematocrit. The signal input to the sensor strip by the measurement
device was a gated amperometric pulse sequence that included eight
pulsed excitations separated by seven relaxations, such as
described in U.S. Patent Publication No. 2008/0173552. The
excitations were less than a 1 second in duration. Three output
current values were recorded during each excitation.
[0062] To correlate the output current values from the input signal
with the analyte concentration of the sample, the initial current
value from the excitation is preferably greater than those that
follow in the decay. The output signals from the sensor strip of
FIG. 6 showed an initial high current value that decays at about
two seconds after the blood sample was introduced to the strip.
Thus, the first output currents having a high initial current value
followed by decaying current values were observed in output current
110.
[0063] To correlate the output current values from the input signal
to the analyte concentration of the sample, different sample
analyte concentrations also preferably show a substantially
constant difference between output signal current values.
Preferably, the output current value or values correlated with the
analyte concentration of the sample also are taken from a decay
including current data reflecting the maximum kinetic performance
of the sensor strip. The kinetics of the redox reaction underlying
the output currents is affected by multiple factors. These factors
may include the rate at which the reagent composition rehydrates,
the rate at which the enzyme system reacts with the analyte, the
rate at which the enzyme system transfers electrons to the
mediator, and the rate at which the mediator transfers electrons to
the electrode. Of these and other kinetic factors affecting the
output currents, the rate at which the reagent composition
rehydrates is believed to have the greatest influence on the output
currents.
[0064] The maximum kinetic performance of the sensor strip may be
reached during an excitation of a gated amperometric pulse sequence
when the initial current value of an excitation having decaying
current values is greatest for the multiple excitations. This may
also be referred to as sensor-peak time. Preferably, the maximum
kinetic performance of a sensor strip is reached when the last in
time current value obtained for an excitation having decaying
current values is the greatest last in time current value obtained
for the multiple excitations. More preferably, the maximum kinetic
performance of a sensor strip is reached when the initial current
value of an excitation having decaying current values is greatest
for the multiple excitations and the last in time current value
obtained for the same excitation is the greatest last in time
current value obtained for the multiple excitations.
[0065] The maximum kinetic performance of the sensor strip is
desirably less than about 3 seconds and even more desirably less
than about 2 seconds.
[0066] The gated amperometric pulse sequence used to determine the
maximum kinetic performance of a test sensor included at least
seven duty cycles, where the excitations are about 0.4 sec. in
duration and the relaxations are 1 sec. in duration, include zero
current flow through the sample, and are provided by an open
circuit. At least three output current values are measured during
each excitation. The potential input to the sensor strip is held
substantially constant, at 250 mV and the sample temperature is at
23.degree. C. Before the duty cycles, a pulse of 400 mV was applied
for 0.9 seconds.
[0067] The sensor strip with 400 mg/dL of glucose in FIG. 6 reached
maximum kinetic performance during the excitation decay that
included output currents 120 and 125, between 3 and 4 seconds from
the introduction of the sample to the sensor strip. This was
established as both the greatest initial and the greatest last in
time current values obtained from an excitation having decaying
current values were present in the cycle that included output
currents 120 and 125. Compare initial output current 120 with
output currents 110, 130, 140, 150, 160 and 170 and also compare
last in time output current 125 with output currents 115, 135, 145,
155, 165 and 175. Thus, the sensor reaches its maximum kinetic
performance in between 3 and 4 seconds even for a 70% hematocrit
blood sample
Example 7
[0068] FIGS. 7-9 show that the reagent formulation can also affect
maximum kinetic performance for blood with different hematocrit
levels. Referring initially to FIG. 7, at a glucose concentration
of 50 mg/dL, the maximum kinetic performance using sensor-peak time
increased as the hematocrit level increased. The mediator that
included a smaller sulfate percentage (5% sulfate) generally had
much faster peak times at comparable hematocrit levels and buffer
concentrations. See, for example, at hematocrit level 60% using 50
mM phosphate buffer (compare 3.5 sec. with mediator using 5%
sulfate and 7.5 sec. with mediator using 20% sulfate). The reagent
formulation, such as buffer strength and residual sulfate content
of the mediator, greatly impacts sensor reaction peak time,
especially for samples with high hematocrit (>40%). Thus, high
inorganic salt (from buffer or from the mediator in terms of
sulfate concentration) in sensor formulation increases sensor-peak
time (i.e., slows down sensor reaction). FIG. 8 shows similar
results using 100 mg/dL of glucose concentration. Using the high
glucose concentration of 400 mg/dL, FIG. 9 shows maximum kinetic
performance using sensor-peak time generally below about 3 or 3.5
seconds with a number of sensor-peak times of 2 seconds. The
mediator with 5% sulfate and 100 mM of phosphate buffer at high
hematocrit levels had a maximum kinetic performance using
sensor-peak time of about 4.5 or 5 seconds.
[0069] Thus, as shown in the 50 mg/dL and 100 mg/dL glucose
concentrations, to achieve a fast reagent re-hydration and glucose
reaction for samples with high hematocrit, the salt content in
reagent formulations has to be lowered.
Example 8
[0070] Two formulations were tested for maximum kinetic performance
after the test sensors had been stored under stressed conditions
(-20.degree. C. and 50.degree. C./4 wks.). The formulation in FIG.
10a included 50 mM phosphate buffer and a
3-(2',5'-disulfophenylimino)-3H-phenothiazine having 5 wt. %
sulfate. The formulation in FIG. 10b included 100 mM phosphate
buffer and a 3-(2',5'-disulfophenylimino)-3H-phenothiazine having
20 wt. % sulfate. The gated amperometric pulse sequence used in
this Example was similar to that described in Example 6 above.
[0071] As shown in FIG. 10a, maximum kinetic performance was about
2 seconds for the samples that were stressed at -20.degree. C. for
4 wks. The maximum kinetic performance was about 3.5 seconds for
the samples that were stressed at -50.degree. C. for 4 wks.
Referring to FIG. 10b, the maximum kinetic performance was about
3.5 seconds for the samples that were stressed at -20.degree. C.
for 4 wks. and about 5 seconds for the samples that were stressed
at -50.degree. C. for 4 wks. Thus, formulations having a lower
inorganic salt amount (e.g., FIG. 10a) had an improved maximum
kinetic performance as compared to formulations with a higher
inorganic salt amount (e.g., FIG. 10b).
[0072] In addition, lots of the formulation used in FIG. 10a with 2
second sensor-peak times were tested for assay bias or % bias. The
% bias for glucose concentrations not greater than 100 mg/dL were
less than about +/-2%. The % bias for a glucose concentration of
400 mg/dL was about +/-4%. The assay biases for lots of formulation
used in FIG. 10b having 3-4 second sensor-peak times were also
tested. The % bias for glucose concentrations not greater than 100
mg/dL were less than about +/-3%. The % bias for a glucose
concentration of 400 mg/dL was about +/-10%. Thus, the assay biases
for the lots having 3-4 second sensor-peak times were greater than
the lots having 2 second sensor-peak times.
Example 9
[0073] Referring to FIG. 11, the coefficient of variation % (% CV)
using 40% hematocrit whole blood samples at different glucose
concentrations are shown. The glucose concentrations range from 36
mg/dL to 627 mg/dL. Reagent solutions having a low salt content lot
were compared to reagent solutions having a high salt content lot.
The low salt lot included 50 mM phosphate buffer at pH 6.5, 2 U/ul
FAD-GDH, 40 mM 3-(2',5'-disulfophenylimino)-3H-phenothiazine having
5 wt. % sulfate, 0.25% hydroxyethyl cellulose-300 k and 0.2% MEGA 8
surfactant. The high salt lot included 100 mM phosphate buffer at
pH 6.5, 2 U/ul FAD-GDH, 40 mM
3-(2',5'-disulfophenylimino)-3H-phenothiazine having 20 wt. %
sulfate, 0.25% hydroxyethyl cellulose-300 k and 0.2% MEGA 8
surfactant.
[0074] The % CV was calculated by taking the mean of the maximum
kinetic performance using sensor-peak times and dividing by the
standard deviation of those sensor-peak times. This resulting value
was multiplied by 100, resulting in the % CV. A total of 40 samples
were tested for both the low salt reagent solutions and the high
salt reagent solutions.
[0075] The low salt reagent solution reached maximum kinetic
performance using sensor-peak times in less than 3 seconds,
resulting in a better % CV for 40% hematocrit whole blood samples
as compared to the higher salt reagent solution. The low salt
reagent solutions had a much better % CV at the lower glucose
concentration samples.
[0076] While the examples provided herein relate to in vitro
applications of the test sensor reagents in accordance with the
present invention, it is contemplated that these reagents may also
be adapted for in vivo analyte monitoring by chemically
immobilizing the mediators (e.g., by chemical reaction at one or
more of the substituent groups on the aromatic rings), and
incorporating the immobilized mediators into a device which can be
implanted subcutaneously into a patient. The reagents of the
embodiments described herein may also be used with continuous
analyte monitoring systems.
Alternative Embodiment A
[0077] A reagent for detecting an analyte, the reagent
comprising:
[0078] a flavoprotein enzyme;
[0079] a mediator selected from the group
##STR00006##
or a combination thereof, wherein R.sup.1, R.sup.2, R.sup.3,
R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8, and R.sup.9 are the
same or different, and are independently selected from the group
consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl,
cyclic, heterocyclic, halo, haloalkyl, carboxy, carboxyalkyl,
alkoxycarbonyl, aryloxycarbonyl, aromatic keto, aliphatic keto,
alkoxy, aryloxy, nitro, dialkylamino, aminoalkyl, sulfo,
dihydroxyboron, and combinations thereof;
[0080] at least one surfactant;
[0081] a polymer; and
[0082] a buffer.
Alternative Embodiment B
[0083] The reagent of Alternative Embodiment A, wherein the
flavoprotein enzyme is FAD-glucose dehydrogenase.
Alternative Embodiment C
[0084] The reagent of Alternative Embodiment A, wherein the
mediator comprises
3-(2',5'-disulfophenylimino)-3H-phenothiazine.
Alternative Embodiment D
[0085] The reagent of Alternative Embodiment A, wherein the
surfactant includes a saccharide-based surfactant or a
phosphorylcholine-based surfactant.
Alternative Embodiment E
[0086] The reagent of Alternative Embodiment A, wherein the polymer
is a cellulose-based polymer.
Alternative Embodiment F
[0087] The reagent of Alternative Embodiment A, wherein the buffer
comprises a phosphate buffer.
Alternative Embodiment G
[0088] A reagent for detecting an analyte in a fluid sample, the
reagent comprising:
[0089] FAD-glucose dehydrogenase having an activity of from about
0.1 Units/.mu.L to about 10 Units/.mu.L;
[0090] a 3-(2',5'-disulfophenylimino)-3H-phenothiazine mediator
having a concentration of from about 5 mM to about 120 mM;
[0091] a heptanoyl-N-methylglucamide surfactant having a
concentration of from about 0.05 wt. % to about 0.5 wt. % of the
reagent; and
[0092] a hydroxyethyl cellulose polymer having a concentration of
from about 0.1 wt. % to about 4 wt. % of the reagent.
Alternative Embodiment H
[0093] The reagent of Alternative Embodiment G further comprising a
phosphate buffer.
Alternative Embodiment I
[0094] The reagent of Alternative Embodiment H, wherein the
phosphate buffer has a concentration of from about 25 mM to about
200 mM and a pH of from about 4 to about 8.
Alternative Embodiment J
[0095] The reagent of Alternative Embodiment I, wherein the
phosphate buffer has a concentration of from about 50 mM to about
150 mM and a pH of from about 6 to about 7.
Alternative Embodiment K
[0096] The reagent of Alternative Embodiment G, wherein the reagent
comprises FAD-glucose dehydrogenase having an activity of from
about 0.5 Units/.mu.L to about 2.5 Units/.mu.L, a
3-(2',5'-disulfophenylimino)-3H-phenothiazine mediator having a
concentration of from about 30 mM to about 60 mM, a
heptanoyl-N-methylglucamide surfactant having a concentration of
from about 0.1 wt. % to about 0.4 wt. % of the reagent, and a
hydroxyethyl cellulose polymer having a concentration of from about
0.2 wt. % to about 0.5 wt. % of the reagent.
Alternative Embodiment L
[0097] An electrochemical test sensor comprising:
[0098] a working electrode having a surface;
[0099] a counter electrode having a surface; and
[0100] a reagent coating at least a portion of the surface of the
working electrode and at least a portion of the surface of the
counter electrode, the reagent comprising a flavoprotein, a
phenothiazine or a phenoxazine mediator, a buffer, and at least one
surfactant and a polymer.
Alternative Embodiment M
[0101] The sensor of Alternative Embodiment L, wherein the
flavoprotein includes FAD-glucose dehydrogenase.
Alternative Embodiment N
[0102] The sensor of Alternative Embodiment L, wherein the
phenothiazine mediator includes
3-(2',5'-disulfophenylimino)-3H-phenothiazine.
Alternative Embodiment O
[0103] The sensor of Alternative Embodiment L, wherein the at least
one surfactant includes a heptanoyl-N-methylglucamide.
Alternative Embodiment P
[0104] The sensor of Alternative Embodiment L, wherein the polymer
is a cellulose-based polymer.
Alternative Process Q
[0105] A method of detecting an analyte in a fluid sample, the
analyte undergoing a chemical reaction, the method comprising the
acts of:
[0106] providing an electrode surface;
[0107] facilitating flow of the fluid sample to the electrode
surface using at least one surfactant;
[0108] catalyzing the chemical reaction with a flavoprotein
enzyme;
[0109] generating a redox equivalent by the chemical reaction;
and
[0110] transferring the redox equivalent to the electrode surface
using a phenothiazine or a phenoxazine mediator.
Alternative Process R
[0111] The method of Alternative Process Q, wherein the electrode
surface includes a working electrode and a counter electrode, the
electrode surface including a reagent comprising the at least one
surfactant, the flavoprotein enzyme, the phenothiazine mediator,
and a buffer.
Alternate Process S
[0112] The method of Alternative Process R, wherein the reagent
further includes a cellulose-based polymer.
Alternate Process T
[0113] The method of Alternative Process S, wherein the polymer is
a cellulose-based polymer.
Alternate Process U
[0114] The method of Alternative Process R, wherein the buffer
includes a phosphate buffer.
[0115] While the invention is susceptible to various modifications
and alternative forms, specific embodiments and methods thereof
have been shown by way of example in the drawings and are described
in detail herein. It should be understood, however, that it is not
intended to limit the invention to the particular forms or methods
disclosed, but, to the contrary, the intention is to cover all
modifications, equivalents, and alternatives falling within the
spirit and scope of the invention.
* * * * *