A REAGENT FORMULATION USING RUTHENIUM HEXAMINE AS A MEDIATOR FOR
ELECTROCHEMICAL TEST STRIPS
1. Priority
[0001[ This application claims the benefits of priority under 35 U.S.C. § 119 from
provisional application S.N. 60/850,221 filed on October 5, 2006, entitled: "A
REAGENT FORMULATION USING A RUTHENIUM BASED MEDIATOR FOR
ELECTROCHEMICAL TEST STRIPS, " which application is incorporated by
reference in its entirety herein.
2. Description of the Related Art
[0002] Electrochemical glucose test strips, such as those used in the OneTouch®
Ultra® whole blood testing kit, which is available from LifeScan, Inc., are designed to
measure the concentration of glucose in a blood sample from patients with diabetes.
The measurement of glucose is based upon the specific oxidation of glucose by the
enzyme glucose oxidase (GO). The reactions which may occur in a glucose test strip
are summarized below in Equations 1 and 2.

[0003] As illustrated in Equation 1, glucose is oxidized to gluconic acid by the
oxidized form of glucose oxidase (GO(OX))- It should be noted that GO(OX) may also be
referred to as an "oxidized enzyme." During the reaction in Equation 1, the oxidized
enzyme GO(0x)is converted to its reduced state which is denoted as GO(red)(ie.,
"reduced enzyme"). Next, the reduced enzyme GO(red)is re-oxidized back to GO(ox) by
reaction with Fc(CN)63- (referred to as either the oxidized mediator or fcrricyanide) as
illustrated in Equation 2. During the re-generation of GO(red)back to its oxidized state

GO(ox), FC(CN)63- is reduced to Fe(CN)64 (referred to as either reduced mediator or
icrrocyanide).
[0004] When the reactions set forth above are conducted with a test voltage applied
between two electrodes, a test current may be created by the electrochemical re-
oxidation of the reduced mediator at the electrode surface. Thus, since, in an ideal
environment, the amount of ferrocyanide created during the chemical reaction
described above is directly proportional to the amount of glucose in the sample
positioned between the electrodes, the test current generated would be proportional to
the glucose content of the sample. A mediator, such as ferricyanide, is a compound
that accepts electrons from an enzyme such as glucose oxidase and then donates the
electrons to an electrode. As the concentration of glucose in the sample increases, the
amount of reduced mediator formed also increases; hence, there is a direct relationship
between the test current, resulting from the re-oxidation of reduced mediator, and
glucose concentration. In particular, the transfer of electrons across the electrical
interface results in the flow of a test current (2 moles of electrons for every mole of
glucose that is oxidized). The test current resulting from the introduction of glucose
may, therefore, be referred to as a glucose current.
(0005[ Because it can be very important to know the concentration of glucose in blood,
particularly in people with diabetes, test meters have been developed using the
principals set forth above to enable the average person to sample and test their blood
for determining their glucose concentration at any given time. The glucose current
generated is detected by the test meter and converted into a glucose concentration
reading using an algorithm that relates the test current to a glucose concentration via a
simple mathematical formula. In general, the test meters work in conjunction with a
disposable test strip that includes a sample receiving chamber and at least two
electrodes disposed within the sample receiving chamber in addition to the enzyme
(e.g. glucose oxidase) and the mediator (e.g. ferricyanide). In use, the user pricks their
finger or other convenient site to induce bleeding and introduces a blood sample to the
sample receiving chamber, thus starting the chemical reaction set forth above.
[0006] In electrochemical terms, the function of the meter is two fold. Firstly, it
provides a polarizing voltage (approximately 400 mV in the case of OneTouch®

Ultra®) that polarizes the electrical interface and allows current (low at the carbon
working electrode surface. Secondly, it measures the current that flows in the external
circuit between the anode (working electrode) and the cathode (reference electrode).
The test meter may, therefore be considered to be a simple electrochemical system that
operates in a two-electrode mode although, in practice, third and, even fourth electrodes
may be used to facilitate the measurement of glucose and/or perform other functions in
the test meter.
[0007[ As previously described, the amount of reduced mediator should be
proportional to the glucose concentration present in a physiological fluid based on the
reactions described in liquation 1 & 2. Under certain circumstances, oxidized
mediators such as, for example, ferricyanide can be converted to fcrrocyanide (reduced
mediator) in the presence of a humid environment. The generation of reduced mediator
by non-glucose dependent reactions may cause a falsely elevated glucose concentration
to be measured which, in turn, affects the assay's accuracy.
[0008] The presence of interfering compounds may also cause the test current to be
falsely elevated because the interfering compound may become oxidized at the working
electrode. Additionally, the oxidized mediator may become reduced by the interfering
compound where the resulting icduced mediator can become oxidized at the working
electrode. One strategy for decreasing the effects of interfering compounds is to use a
relatively low test voltage between the working electrode and reference electrode. In
order to employ a lower test voltage, the reagent formulation requires the mediator to
have a lower redox voltage.
[0009] As such, applicants recognize that there is a great need for using mediators
which do not convert to the reduced state in the presence of humidity and have a
relatively low redox voltage. Further, such mediators need to be incorporated into a
reagent formulation that can be easily coated onto a test strip in a robust manner
enabling accurate and precise glucose measurements.

SUMMARY OF INVENTION
[0010] In one embodiment, a test strip is provided which is capable of measuring an
analyte. The test strip may include a working electrode and a reference electrode where
the reagent formulation is disposed on the working electrode. The reagent formulation
may be coated onto the test strip. 'Hie reagent formulation includes an enzyme, a
ruthenium hexamine mediator, and a solution for dissolving the enzyme and the
ruthenium hexamine mediator. The ruthenium hexamine has a concentration range
from about 15% to about 20% (weight of mediator / volume) of solution. The enzyme
may be either glucose oxidase and glucose dehydrogenase. When using glucose
oxidase, the enzyme activity per unit volume may range from about 1500 units/mL to
about 8000units/mL. The solution for dissolving the enzyme may be a buffer such as,
for example, phosphate, citrate, or citraconate. When using phosphate buffer, the pH
may be about 7.
[0011 ] In another embodiment, the reagent formulation may further include a filler
having hydrophilic and hydrophobic domains. In one embodiment, the filler may be a
silica such as for example Cab-o-Sil TS 610. The formulation may be disposed on the
working electrode using a process of screen printing. The screen may have a frame
which secures a plurality of interwoven threads. The plurality of interwoven threads
may form a plurality of open rectangular spaces for allowing the reagent formulation to
pass therethrough. The plurality of interwoven threads may have a thread spacing and
a thread diameter. The thread spacing may range from about 90 threads per centimeter
to about 120 threads per centimeter. The thread diameter may range from about 30
microns to about 50 microns.
[0012] In another aspect, a test strip includes a substrate, two electrodes and a reagent.
The generally planar substrate extends from a first end to a second end. The first and
second electrodes are disposed on the substrate proximate one of the first and second
ends with a reagent layer disposed thereon. The reagent layer has ruthenium hexamine
trichloride in a buffer solution with a concentration range from about 15% to about
20% (weight / volume) such that the test strip shows essentially no increase in bias to
reference when a blood sample is tested at about 400 millivolts after storage for seven
days at 40 degrees Celsius at 75% relative humidity.

[00131 hi a further aspect, a test strip is provided that includes a substrate, two
electrodes and a reagent. The generally planar substrate extends from a first end to a
second end. The first and second electrodes arc disposed on the substrate proximate
one of the first and second ends with a reagent layer disposed thereon. The reagent
layer has a reagent layer with ruthenium hexamine Trichloride in a buffer solution with
a concentration range from about 15% to about 20% (weight / volume) such that the
test strip shows essentially no increase in bias to reference when tested at about 400
millivolts with blood glucose sample of about 70 mg/dL with uric acid concentration of
about 0 mg/dL to about 20 mg/dL.
[0014] In a further aspect, a test strip is provided that includes a substrate, two
electrodes and a reagent. The generally planar substrate extends from a first end to a
second end. The first and second electrodes are disposed on the substrate proximate
one of the first and second ends with a reagent layer disposed thereon. The reagent
layer has a reagent layer with an enzyme and ruthenium hexamine trichloride in a
buffer solution with a concentration range from about 15% to about 20% (weight /
volume) such that the test strip shows essentially no increase in bias to reference when
tested at about 400 millivolts with blood glucose sample of about 70 mg/dL with
gentisic acid concentration from about 0 mg/dL to about 50 mg/dL.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The novel features of the invention are set forth with particularity in the
appended claims. A better understanding of the features and advantages of the present
invention will be obtained by reference to the following detailed description that sets
forth illustrative embodiments, in which the principles of the invention arc utilized, and
the accompanying drawings of which:
[16] Figure 1 illustrates a top exploded perspective view of a prior art embodiment
of an unassembled test strip;
[17] Figure 2 illustrates a top plan view of the prior art test strip as shown in Figure 1
after it has been assembled;

[0018] Figure 3 illustrates a top plan view of a test meter connected to the prior art test
strip as shown in Figures 1 and 2;
[0019] Figure 4 illustrates a simplified schematic view of the test meter of Figure 3
forming an electrical connection with the test strip of Figures 1 and 2;
[0020] Figure 5a is a graph illustrating the application of an applied test voltage, from
the test meter of Figure 3, to the test strip of Figures 1 and 2, for a test time interval tr
for generating a test current which can be used for calculating an analyte concentration;
[0021] Figure 5b is a graph illustrating an alternative embodiment for applying test
voltages and an open-circuit time interval, from the test meter of Figure 3, to the test
strip of Figures I and 2, for a test time interval tr for generating a test current which
can be used for calculating an analyte concentration;
[0022] Figure 5c is an expanded view of the graph of Figure 5b illustrating a serial
application of an applied test voltage for detecting fluid, an open-circuit time interval,
and another applied test voltage for measuring an analyte concentration;
[0023] Figure 6 is a graph illustrating a test current which results from the applied test
voltage of Figure 5a when a blood sample is applied to the test strip of Figures 1 and 2;
[0024] Figure 7 illustrates a top exploded perspective view of an unassembled test strip
in another embodiment;
[0025] Figure 8 is a graph showing the average bias to reference measurement of a test
strip measurement for test strips using a ferricyanide based reagent layer stored for
seven days at room temperature in a desiccated environment (filled in circles) or at 40
°C in a 75% relative humidity (RH) (open squares),
[0026] Figure 9 is a graph showing the average bias to reference measurement of a test
strip measurement for test strips using a ruthenium based reagent layer stored for seven
days at room temperature in a desiccated environment (filled in circles) or at 40 °C in a
75% relative humidity (RH) (open squares);
[0027] Figure 10 is a graph showing the average bias to reference measurement of a
test strip measurement for test strips using a using either ferricyanide based reagent
layer (open triangles) or a ruthenium based reagent layer (filled in diamonds) where the
blood samples had varying concentrations of uric acid; and

[0028[ Figure 11 is a graph showing the average bias to reference measurement of a
test strip measurement for test strips using a using either ferricyanide based reagent
layer (open triangles) or a ruthenium based reagent layer (filled in diamonds) where the
blood samples had varying concentrations of gentisic acid.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS OF THE
INVENTION
[0029] Embodiments of the present invention are directed to a reagent formulation for
use in electrochemical test strips. In particular, the embodiments include the use of"
ruthenium hexamine as a mediator to enable the manufacture of test strips which have
increased stability under high humidity environmental conditions and/or also have
decreased oxidation of interfering compounds. In an embodiment, a reagent
formulation is described which may be easily coated onto a test strip in a robust manner
enabling accurate and precise glucose measurements.
[0030] Figure 1 is an exploded perspective view of a prior art test strip 100, which
includes six layers disposed on a substrate 5. These six layers may be a conductive
layer 50, an insulation layer 16, a reagent layer 22, an adhesive layer 60, a hydrophilic
layer 70, and a top layer 80. Test strip 100 may be manufactured in a series of steps
wherein the conductive layer 50, insulation layer 16, reagent layer 22, adhesive layer
60 are sequentially deposited on substrate 5 using, for example, a screen printing
process as described in U.S. Pre-Grant Publication No. US20050096409A1 and
published International Application No.'s WO2004040948A1, WO2004040290A1,
WO2004040287A1, WO2004040285A2, WO2004040005A1, WO2004039897A2, and
WO2004039600A2. In an alternative embodiment, an ink jetting process may be used
to deposit reagent layer 22 which is described in U.S. Patent No. 6,179,979.
Hydrophilic layer 70 and top layer 80 may be disposed from a roll stock and laminated
onto substrate 5. Test strip 100 has a distal portion 3 and a proximal portion 4 as
shown in Figures 1 and 2.

[0031) The fully assembled lest strip 100, as shown in Figure 2, includes an inlet 90
through which a blood sample may be drawn into a sample receiving chamber 92. Inlet
90 may be formed by cutting through a distal portion 3 of test strip 100. A blood
sample 94 can be applied to inlet 90, as illustrated in Figure 3, to fill a sample receiving
chamber 92 so that glucose can be measured. The side edges of a first adhesive pad 24
and a second adhesive pad 26 located adjacent to reagent layer 22 each define a wall of
sample receiving chamber 92. A bottom portion or "'floor" of sample receiving
chamber 92 includes a portion of substrate 5, conductive layer 50, and insulation layer
16. A top portion or "roof" of sample receiving chamber 92 includes distal hydrophilic
portion 32.
[0032] For test strip 100, as illustrated in Figure 1, conductive layer 50 includes a
reference electrode 10, a first working electrode 12, a second working electrode 14, a
first contact 13, a second contact 15, a reference contact 11, a first working electrode
track 8, a second working electrode track 9, a reference electrode track 7, and a strip
detection bar 17. The conductive layer may be a carbon ink such as the one described
in U.S. Patent No. 5,653,918. First contact 13, second contact 15, and reference
contact 1 1 may be adapted to electrically connect to test meter 200. First working
electrode track 8 provides an electrically continuous pathway from first working
electrode 12 to first contact 13. Similarly, second working electrode track 9 provides
an electrically continuous pathway from second working electrode 14 to second contact
15. Similarly, reference electrode track 7 provides an electrically continuous pathway
from reference electrode 10 to reference contact 1 1.
[0033] In Figure 1, insulation layer 16 includes an aperture 18 which exposes a portion
of reference electrode 10, first working electrode 12, and second working electrode 14
which can be wetted by a liquid sample. As an example, insulation layer 16 may be
Ercon E6110-116 Jet Black Insulayer™ ink which may be purchased from F.rcon, Inc
(Waltham, Massachusetts).
[0034] Reagent layer 22 may be disposed on a portion of conductive layer 50, substrate
5, and insulation layer 16 as illustrated in Figure 1. Reagent layer 22 may include
chemicals such as an enzyme and a mediator which selectivity reacts with glucose. An

example of an enzyme may be glucose oxidase and an example of a mediator may be
ferricyanidc.
[0035] Examples of enzymes suitable for use in this invention may include either
glucose oxidase or glucose dehydrogenase. More specifically, the glucose
dehydrogenase may have a pyrrylo-quinoline quinone co-factor (abbreviated as PQQ or
may be referred to its common name which is methoxatin). Examples of mediator
suitable for use in this invention may include either ferricyanidc or ruthenium
hexamine trichloride ([RuIII(NII3)6]Cl3 and may also be simply referred to as ruthenium
hexamine). During the reactions as illustrated in liquations 1 and 2, a proportional
amount of reduced mediator can be generated that is electrochemically measured for
calculating a glucose concentration. Examples of reagent formulations or inks suitable
for use in the embodiments can be found in US patents 5,708,247 6,046,051, and
6,241,862; U.S. Pre-Grant Publication No. 20030217918A1; published international
applications WOO 1/67099 and WO01/73124.
[0036] Reagent layer 22 may be formed from an enzyme ink or formulation, which is
disposed onto a conductive layer and dried. An enzyme ink or formulation typically
contains a liquid, such as a buffer, for dispersing and/or dissolving materials used for
the electrochemical detection of an analytc such as glucose. Buffers which may be
suitable for the formulation can be phosphate, citrate and citraconate.
[0037] In an embodiment, the formulation may include a 200 mM phosphate buffer
having a pH of about 7 and a ruthenium hexamine mediator concentration ranging from
about 5% and greater, preferably ranging from about 10% and greater, and yet more
preferably ranging from about 15% to about 20% (percentage based on weight of
mediator / volume of buffer). The pH of around 7 was chosen because glucose oxidase
has a sufficiently high activity at this pH when using ruthenium hexamine as a
mediator. The upper range for the ruthenium hexamine concentration may be selected
based on its solubility. When the enzyme ink is formulated to have greater than a 20%
ruthenium hexamine concentration, solid particles of ruthenium hexamine may be
present in reagent layer 22 which do not dissolve during testing. The presence of
undissolved ruthenium hexamine may cause a decrease in the test strip-to-test strip
precision. When the enzyme ink is formulated to have less than a 15% ruthenium

hcxaminc concentration, the magnitude of the test current values may decrease with the
concentration of ruthenium hexamine. In general, it is undesirable for the magnitude of
the test current values to be dependent on the concentration of ruthenium hexamine
because small changes in ruthenium hexamine concentration will cause variability in
the test current values and., in turn, the strip lot-to-strip lot variability.
[0038] In an embodiment, the formulation may have an enzyme activity ranging from
about 1500 units/mL to about 8000 units/mL. The enzyme activity range may be
selected so that the glucose current does not depend on the level of enzyme activity in
the formulation so long as the enzyme activity level is within the above stated range.
The enzyme activity should be sufficiently large to ensure that the resulting glucose
current will not be dependent on smaJl variations in the enzyme activity. For instance,
the glucose current will depend on the amount of enzyme activity in the formulation if
the enzyme activity is less than 1500 units/mL. On the other hand, for enzyme activity
levels greater than 8000 units/mL, solubility issues may arise where the glucose
oxidase cannot be sufficiently dissolved in the formulation. Glucose oxidase may be
commercially available from Biozymc Laboratories International Limited (San Diego,
California, U.S.A.). The glucose oxidase may have an enzyme activity of about 250
units/mg using where the enzyme activity units are based on an o-dianisidine assay at
pM 7 and 25 "C.
[0039] An enzyme ink which contains a filler having both hydrophobic and hydrophilic
domains may be disposed onto the working electrode using a screen printing process.
An example of a filler may be a silica such as, for example, Cab-o-Sil TS 610 which is
commercially available from Cabot Inc., Boston, Massachusetts. Typically, a screen
may be in the form of a rectangular frame which secures a plurality of interwoven
threads. The plurality of interwoven threads form a plurality of open rectangular
spaces for allowing enzyme ink to pass therethrough. The density and the size of the
open spaces influence the amount of enzyme ink which becomes deposited on the
conductive layer. Characteristics of the interwoven threads which influence the
deposition of the enzyme ink are thread spacing and thread diameter. The thread
spacing may range from about 90 threads per centimeter to about 120 threads per
centimeter. The thread diameter may range from about 30 microns to about 50

microns. More specifically, in an embodiment, a screen suitable for screen printing an
enzyme ink having ruthenium hexamine and glucose oxidase may have a thread
spacing of about 120 threads per centimeter and a thread diameter of about 34 microns.
[0040] For test strip 100, adhesive layer 60 includes first adhesive pad 24, second
adhesive pad 26, and third adhesive pad 28 as illustrated in Figure 1. Adhesive layer
60 may include a water based acrylic copolymer pressure sensitive adhesive which is
commercially available from Tape Specialties LTD which is located in Tring, Herts,
United Kingdom (part#A6435). Adhesive layer 60 is disposed on a portion of
insulation layer 16, conductive layer 50, and substrate 5. Adhesive layer 60 binds
hydrophilic layer 70 to test strip 100.
[0041] Hydrophilic layer 70 includes a distal hydrophilic portion 32 and proximal
hydrophilic portion 34. Hydrophilic layer 70 may be a polyester having one
hydrophilic surface such as an anti-fog coating which is commercially available from
3M.
[0042] For test strip 100, top layer 80 includes a clear portion 36 and opaque portion 38
as illustrated in Figure I. Top layer 80 is disposed on and adhered to hydrophilic layer
70. Top layer 80 may be a polyester. It should be noted that the clear portion 36
substantially overlaps distal hydrophilic portion 32 which allows a user to visually
confirm that the sample receiving chamber 92 may be sufficiently filled. Opaque
portion 38 helps the user observe a high degree of contrast between a colored fluid such
as, for example, blood within the sample receiving chamber 92 and the opaque portion
38 of top layer 80.
[0043] Figure 3 illustrates a test meter 200 suitable for connecting to test strip 100.
Test meter 200 includes a display 202, a housing 204, a plurality of user interface
buttons 206, and a strip port connector 208. Test meter 200 further includes electronic
circuitry within housing 204 such as a memory 210, a microprocessor 212, electronic
components for applying a test voltage, and also for measuring a plurality of test
current values (see 104 and 106 in Figure 4). Proximal portion 4 of test strip 100 may
be inserted into strip port connector 208. Display 202 may output a glucose
concentration and also may be used to show a user interface for prompting a user on

how to perform a test. The plurality of user interface buttons 206 allow a user to
operate test meter 200 by navigating through the user interface software.
[0044] Figure 4 shows a simplified schematic of test meter 200 interfacing with test
strip 100. Test meter 200 includes a first connector 103, second connector 102, and a
reference connector 101 which respectively form an electrical connection to first
contact 13, second contact 15, and reference contact 11: The three aforementioned
connectors are part of strip port connector 208. When performing a test, a first test
voltage source 104 applies a first test voltage VI between first working electrode 12 and
reference electrode 10. As a result of first test voltage VI , test meter 200 may then
measure a first test current II In a similar manner, second test voltage source 106
applies a second test voltage V2 between second working electrode 14 and reference
electrode 10. As a result of second test voltage V2 test meter 200 may then measure a
second test current I2. In an embodiment, first test voltage VI and second test voltage
V2 may be about equal allowing a glucose measurement to be performed twice where a
first measurement is performed with first working electrode 12 and a second
measurement is performed with second working electrode 14. The use of two glucose
measurements can increase accuracy by averaging the two results together. For
simplifying the description of the following sections, the algorithms for determining an
accurate glucose concentration will be described for only one working electrode and
reference electrode. It should be apparent to one skilled in the art, that the invention
should not be limited to one working electrode and reference electrode, but that
multiple working electrodes can also be applied to the embodiments.
[0045] Figure 5a is a chart showing a test voltage that would be applied by test meter
200 to test strip 100 for a test time interval tT which starts when physiological fluid is
detected by test strip 100. In Figure 5a, the test voltage shown is 400 mV. As
illustrated in Figure 5a, before the physiological fluid is applied, test meter 200 is in a
fluid detection mode in which a fluid detection voltage may be 400 mV. It will be
apparent to one skilled in the art that the test voltage and the fluid detection voltage can
be different. In Figure 5a, the test meter is in a fluid detection mode during fluid
detection time interval tFD prior to the detection of physiological fluid at time to- In the
fluid detection mode, test meter 200 determines when a fluid is applied to inlet 90 and

pulled into sample receiving chamber 92 such that the fluid wets both first working
electrode 12 and reference electrode 10. Note that first working electrode 12 and
reference electrode 10 are effectively short-circuited when the physiological fluid
contiguously covers both first working electrode 12 and reference electrode 10. Once
test meter 200 recognizes that the physiological fluid has been applied because of, for
example, a sufficient increase in the measured test current, test meter 200 assigns a zero
second marker at time t0 and starts the test time interval tT For example, as shown in
Figure 5a, test time interval tT is about 5.4 seconds. Upon the completion of the test
time interval tT, the test voltage is removed.
[0046] In an alternative embodiment, a test voltage waveform can be applied that
includes an open-circuit time interval after fluid is detected in sample receiving
chamber 92. For example, the test voltage as shown in Figure 5b is 400 raV during a
fluid detection time interval tFD. Once test meter 200 recognizes that the physiological
fluid has been applied because, for example, the lest current has increased to a level
greater than a pre-determined threshold (e.g., about 50 nanoamperes) for a pre-
determined time interval (e.g., about 20 milliseconds), test meter 200 assigns a zero
second marker at time t0,and applies an open-circuit for an open-circuit time interval
tOC. For example, as shown in Figure 5c, the open-circuit time interval tOC is about 300
milliseconds. Upon completion of the open-circuit time interval tOC, the test meter
applies a test voltage of about 250 millivolts for a time interval ranging from about 200
milliseconds to about 5.4 seconds.
[0047] In general, it is desirable to use a test voltage which is more positive than a
redox voltage of the mediator used in the test strip. In particular, the test voltage
should exceed the redox voltage by an amount sufficient to ensure that the resulting test
current will not be dependent on small variations in the test voltage. Note that a redox
voltage describes a mediator's intrinsic affinity to accept or donate electrons when
sufficiently close to an electrode having a nominal voltage. When a test voltage is
sufficiently positive with respect to the mediator's redox voltage, the mediator will be
rapidly oxidized. In fact, the mediator will be oxidized so quickly at a sufficiently
positive test voltage (i.e., limiting test voltage) that the test current magnitude will be
limited by the diffusion of the mediator to the electrode surface (i.e., limiting test

current). For an embodiment where first working electrode 12 is a carbon ink and the
mediator is ferricyanide, a test voltage of about +400 mV may be sufficient to act as a
limiting test voltage. For an embodiment where first working electrode 12 is a carbon
ink and the mediator is Ru'"(NH3)6, a test voltage of about + 250 mV may be sufficient
to act as a limiting test voltage. It will be apparent to one skilled in the art that other
mediator and electrode material combinations will require different limiting test
voltages.
[0048[ A test meter that is designed to apply a limiting test voltage can have some
variation in the applied test voltage without affecting the magnitude of the limiting test
current. It is desirable for a test meter to apply a limiting test voltage because the test
meter can be constructed with relatively inexpensive electronic components because it
is not necessary to tightly control the test voltage. In summary, a test meter which
applies a limiting test voltage can robustly measure a glucose concentration in an
accurate and precise manner using low cost components.
[0049] Figure 6 is a chart showing the test current generated by test strip 100 during
test time interval tT. hi general, the test current increases rapidly when test strip 100 is
initially wetted with the physiological fluid and then forms a peak at a maximum peak
time tp. After the peak maximum, the test current gradually decreases. The overall
magnitude of the test currents will increase with increasing glucose concentration.
[0050] To produce sufficiently accurate and precise glucose concentration, test strip
100 should have a sufficiently reproducible and well defined electroactive area for first
working electrode 12 and second working electrode 14. The magnitude of the test
current is directly proportional to the area of one of the working electrodes. For
instance, if the variation in the area of first working electrode 12 was high from one test
strip to another, then the variation observed in the measured glucose concentrations will
also be high when testing multiple test strips. Thus, it is important that the variation for
first working electrode 12 and second working electrode 14 be relatively small so that
the variation in the measured glucose concentration can, in turn, also be relatively
small.
[0051 ] The area of first working electrode 12 and second working electrode 14, as
shown in Figures 1 and 2, may be defined by the process of screen printing. A batch of

test strips made by the process of screen printing can output sufficiently precise glucose
concentration measurements (e.g., less than about a 5% CV). However, under certain
circumstances and conditions, there may be a need to manufacture test strip batches
which have even more precise electroactive areas. For instance, there has been a
customer driven desire to design test strips which can measure glucose using very small
volumes of blood (e.g., < 1 microliter). One of the theories being that the use of less
blood will result in less pain during the glucose measuring procedure.
[0052] One strategy for designing test strips which have smaller sample volumes is to
decrease the area of the first and second working electrode. However, as first and
second working electrode and second working electrode become smaller, non-idealThes
in the screen printing process may start to significantly affect the precision of the
working electrodes. For instance, variable stretching of a screen by a squeegee may
affect the size of a working electrode. Additionally, a screen typically has a plurality of
rectangular openings for deposing either a conductive layer or an insulation layer. The
plurality of rectangular opening may cause an edge of either the conductive layer or
insulation layer to be jagged especially when the si/.e of the working electrode is
comparable to the size of the rectangular opening. In the prior art embodiment as
illustrated in Figures 1 and 2, first working electrode 12 has two sides 20 as defined by
the screen printed conductive layer 50 and the other two sides 19 as defined by the
screen printed insulation layer 16. It should be noted that insulation layer 16 is used to
help cover the first working electrode track 9. Thus, there is a need to develop a
process for manufacturing test strips which can have a more precise electrode areas that
do not suffer from the disadvantages of screen printing. Further, there is a need to
develop simpler manufacturing process which do not need an insulation layer for
defining the electrode area and/or for covering the working electrode track.
[0053] In an embodiment, a test strip may be manufactured using a process of laser
ablation for improving the accuracy and precision of the electroactive area of the first
and second working electrode. The process of laser ablation on a conductive layer
allows the edge definition of the electrode area to be better controlled than with other
processes such as screen printing. In addition, the process of laser ablation may be
used to substantially define the electrode area without the need of an insulation layer.

[00541 Figure 7 illustrates a top exploded perspective view of an unassembled test strip
500, which is an embodiment that can also utilize the previously described reagent
formulation. Similar to test strip 100, test strip 500 includes a conductive layer 501, a
reagent layer 570, and a top tape 81. Test strip 500 has a distal portion 576, a proximal
portion 578, and two sides 574.
[0055] Although various embodiments described herein are particularly adapted to the
measurement of a glucose concentration in blood, it will be apparent to those skilled in
the art that the test strip described herein may be adapted to enable an improved
precision for the electrochemical measurement of other analytes. Examples of other
analytes that may be measured with the test strip embodiments are lactate, cthanol,
cholesterol, amino acids, choline, hemoglobin, and fructosamine in blood.
EXAMPLE 1
[0056] The reagent layer was formulated as an enzyme ink suitable for screen printing
as follows. 100 ml of 200 mM aqueous phosphate buffer was adjusted to pH 7. A
mixture was formed by adding 5 g of hydroxyethyl cellulose (HEC), 1 g of poly(vinyl
pyrrolidone vinyl acetate) (PVP-VA S-630), 0.5 ml of DC 1500 Dow Corning antifoam
to 100 ml. of phosphate buffer and mixed by homogenization. The mixture was
allowed to stand overnight to allow air bubbles to disperse and then used as a stock
solution for the formulation of the enzyme ink. Next, 7.5 grams of Cab-o-Sil TS610
was gradually added by hand to the mixture until about 4/5 of the total amount of Cab-
o-Sil TS610 had been added. The remainder Cab-o-Sil TS6I0 was added with mixing
by homogenization. The mixture was then rolled for 12 hours. About 18 g of
ruthenium hexamine ([RuIII(NH3)6]Cl3) was then added and mixed by homogenization
until dissolved. Finally, 2.8 g of glucose oxidase enzyme preparation (250 Units/mg)
was added and then thoroughly mixed into the solution. The resulting formulation was
ready for printing, or could be stored with refrigeration.
EXAMPLE 2

[0057] A first batch of test strips 100 was prepared using a ruthenium based reagent
formulation as described in Example 1. A second batch of test strips 100 was prepared
with a reagent formulation using a ferricyanide mediator instead of ruthenium
hexamine in a manner similar to Example 1. A portion of the first and second batch of
test strips 100 were stored at room temperature in a desiccated environment for 7 days.
Another portion of the first and second batch of test strips 100 were stored at 40 °C in a
70% relative humidity environment for 7 days.
[0058] The first and second batch of test strips were tested in a test meter using a test
voltage at t-400 mV. Blood samples were tested which had a glucose concentration
ranging from about 70 mg/dL to about 600 mg/dL.
[0059] Figure 8 is a graph showing that average bias to reference measurement for the
second batch of test strips had a positive bias to reference measurement when they were
stored for seven days at 40 °C in a 75% relative humidity (RH) (open squares). In
general, the positive bias to reference measurement was the largest at low glucose
concentration which in this case was about a 60% bias to reference measurement at
about 70 mg/dL glucose concentration. In contrast, the second batch of test strips
which was stored at room temperature for seven days in a desiccated environment
showed essentially no increase in bias to reference measurement, as illustrated by the
filled in circles on Figure 8. Thus, test strips having ferricyanide which are exposed to
relatively high humidity show an increase in bias to reference measurement.
[0060] Figure 9 is a graph showing that average bias to reference measurement for the
first batch of test strips that were stored for seven days at 40 °C in a 75% relative
humidity (RU) (open squares) or at room temperature in a desiccated environment
(filled in circles) showed no overall increase in bias to reference measurement. Thus,
test strips having ruthenium which are exposed to relatively high humidity show no
increase in bias to reference measurement which is in contrast to test strip having
ferricyanide.
EXAMPLE 3
[0061] The first and second batch of test strips (as described in Example 2) were tested
in a test meter using a test voltage at +400 mV. Blood samples were tested which had a

glucose concentration of about 70 mg/dL and a uric acid concentration ranging from
about 0 mg/dL to about 20 mg/dL.
[0062] For the second batch of test strips as illustrated by open triangles in Figure 10,
the average bias to reference measurement of the test strip measurements increased
with increasing amounts of uric acid in an approximately linear manner. Therefore, test
strips using a ferricyanide based reagent layer showed that uric acid, which is an
interfering compound, can be oxidized by ferricyanide creating a non-glucose related
increase in the test current.
[0063] For the first batch of test strips as illustrated by filled in diamonds in Figure 10,
the average bias to reference measurement of the test strip measurements did not
increase with increasing amounts of uric acid. Therefore, test strips using a ruthenium
based reagent layer showed that uric acid, which is a potentially interfering compound,
was not oxidized by ruthenium enabling a more glucose selective measurement.
EXAMPLE 4
[0064] The first and second batch of test strips (as described in Example 2) were tested
in a test meter using a test voltage at +400 mV. Blood samples were tested which had a
glucose concentration of about 70 mg/dL and a gentisic acid concentration ranging
from about 0 mg/dL to about 50 mg/dL.
[0065] For the second batch of test strips as illustrated by open triangles in Figure 11,
the average bias to reference measurement of the test strip measurements increased
with increasing amounts of gentisic acid in an approximately linear manner. Therefore,
test strips using a ferricyanide based reagent layer showed that gentisic acid, which is
an interfering compound, can be oxidized by ferricyanide creating a non-glucose
related increase in the lest current.
[0066] For the first batch of test strips as illustrated by filled in diamonds in Figure 11,
the average bias to reference measurement of the test strip measurements did not
increase with increasing amounts of gentisic acid. Therefore, test strips using a
ruthenium based reagent layer showed that gentisic acid, which is a potentially
interfering compound, was not oxidized by ruthenium enabling a more glucose
selective measurement.

[0067] While the invention has been described in terms of particular variations and
illustrative figures, those of ordinary skill in the art will recognize that the invention is
not limited to the variations or figures described. In addition, where methods and steps
described above indicate certain events occurring in certain order, it is intended that
certain steps do not have to be performed in the order described but in any order as long
as the steps allow the embodiments to function for their intended purposes. Therefore,
to the extent there are variations of the invention, which are within the spirit of the
disclosure or equivalent to the inventions found in the claims, it is the intent that this
patent will cover those variations as well.

CLAIMS
WHAT IS CLAIMED IS:
1. A formulation for coating a test strip, the formulation comprising:
an enzyme;
a ruthenium hexamine mediator; and
a solution that dissolves the enzyme and the ruthenium hexamine mediator and in
which the ruthenium hexamine comprises a concentration range from about 15% to
about 20% of weight.
2. The formulation of Claim 1, wherein the enzyme is a material selected from the group
consisting of essentially glucose oxidase and glucose dehydrogenase.
3. The formulation of Claim 1, wherein the enzyme is glucose oxidase and the glucose oxidase
has an activity range from about 1500 units/mL to about 8000 units/mL.
4. The formulation of Claim 1, wherein the solution is a buffer selected from the group
consisting of phosphate, citrate, and citraconate.
5. The formulation of Claim 4, wherein the buffer is phosphate and has a pH of about 7.
6. The formulation of Claim 1, wherein the test strip comprises a working electrode and a
reference electrode, and the formulation is disposed on the working electrode
7. The formulation of Claim 1, wherein the formulation further comprises a filler having
hydrophilic and hydrophobic domains.
8. The formulation of Claim 7, wherein the filler comprises silica.
9. The formulation of Claim 7, wherein the formulation is disposed on the working electrode
using a process of screen printing, wherein the screen has a plurality of interwoven threads, the

plurality of interwoven threads forming a plurality of open rectangular spaces for allowing the
formulation to pass therethrough, the plurality of interwoven threads having a thread spacing
and a thread diameter, wherein the thread spacing ranges from about 90 threads per centimeter
to about 120 threads per centimeter and the thread diameter may range from about 30 microns
to about 50 microns.
10. A test strip comprising:
a generally planar substrate that extends from a first end to a second end;
first and second electrodes disposed on the substrate proximate one of the first and
second ends; and
a reagent layer having ruthenium hexamine trichloride in a buffer solution with a
concentration range from about 15% to about 20% (weight / volume) such that the test strip
shows essentially no increase in bias to reference when a blood sample is tested at about 400
millivolts after storage for seven days at 40 degrees Celsius at 75% relative humidity.
11. A test strip comprising:
a generally planar substrate that extends from a first end to a second end;
first and second electrodes disposed on the substrate proximate one of the first and
second ends; and
a reagent layer having ruthenium hexamine Trichloride in a buffer solution with a
concentration range from about 15% to about 20% (weight / volume) such that the test strip
shows essentially no increase in bias to reference when tested at about 400 millivolts with
blood glucose sample of about 70 mg/dL with uric acid concentration of about 0 mg/dL to
about 20 mg/dL.
12. A test strip comprising:
a generally planar substrate that extends from a first end to a second end;
first and second electrodes disposed on the substrate proximate one of the first and
second ends; and
a reagent layer having an enzyme and ruthenium hexamine Trichloride in a buffer
solution with a concentration range from about 15% to about 20% (weight / volume) such that

the test strip shows essentially no increase in bias to reference when tested at about 400
millivolts with blood glucose sample of about 70 mg/dL with gentisic acid concentration from
about 0 mg/dL to about 50 mg/dL.
13. The test strip of claim 12, in which the enzyme is a material selected from the group
consisting of essentially glucose oxidase and glucose dehydrogenase.
14. The test strip of claim 12, in which the enzyme is glucose oxidase and the glucose oxidase
has an activity range from about 1500 units/mL to about 8000 units/mL.
15. The test strip of claim 12, in which the buffer is selected from the group consisting of
phosphate, citrate, and citraconate.
16. The test strip of Claim 15, wherein the buffer is phosphate and has a pH of about 7.
17. The test strip of Claim 12, wherein the test strip comprises a working electrode and a
reference electrode, and the reagent layer is disposed on the working electrode
18. The test strip of Claim 12, wherein the reagent layer further comprises a filler having
hydrophilic and hydrophobic domains.
19. The test strip of Claim 18, wherein the filler comprises a silica.

Described herein are various embodiments of a test strip, which may be capable of measuring an analyte. The test strip may include a working electrode and a reference electrode where the reagent formulation is disposed on the working electrode. The reagent formulation may be coated onto the test strip. The reagent formulation includes an enzyme, a ruthenium hexamine mediator, and a solution for dissolving the
enzyme and the ruthenium hexamine mediator. The reagent formulation may be coated onto the test strip. The reagent formulation includes an enzyme, a ruthenium hexamine mediator, and a solution for dissolving the enzyme and the ruthenium hexamine mediator. The ruthenium hexamine has a concentration range from about
15% to about 20% (weight of mediator / volume) of
solution. The enzyme may be either glucose oxidase and glucose dehydrogenase.