USOO5951836A

United States Patent 19 11 Patent Number: 5,951,836

McAleer et al. (45) Date of Patent: *Sep. 14, 1999

54) DISPOSABLE GLUCOSE TEST STRIP AND 56) References Cited

METHOD AND COMPOSITIONS FOR
MAKING SAME U.S. PATENT DOCUMENTS
75 Inventors: Jerome F. McAleer, Wantage; David 5,708,247 1/1998 McAleer et al. ........................ 204/403
Scott, Witney; Geoff Hall, Inverness;
Manuel Alvarez-Icaza, InverneSS, Primary Examiner Bruce F. Bell
Elliot V. Plotkin, Inverness, all of Attorney, Agent, or Firm-Oppedahl & Larson LLP
United Kingdom
57 ABSTRACT
73 Assignee: Selfcare, Inc., Waltham, Mass.
An improved disposable glucose test Strip for use in a test
* Notice: This patent is Subject to a terminal dis- meter of the type which receives a disposable test Strip and
claimer. a Sample of blood from a patient and performs an electro
chemical analysis is made using a working formulation
21 Appl. No.: 09/005,710 containing a filler, an enzyme effective to oxidize glucose,
e.g., glucose oxidase, and a mediator effective to transfer
22 Filed: Jan. 12, 1998 electrons from the enzyme. The working formulation is
printed over a conductive carbon base layer to form a
Related U.S. Application Data working electrode. The filler, for example a Silica filler, is
selected to have a balance of hydrophobicity and hydrophi
63 Continuation of application No. 08/601.223, Feb. 14, 1996, licity Such that one drying it forms a two-dimensional
Pat. No. 5,708.247. network on the surface of the conductive base layer. The
(51) Int. Cl." ..................................................... G01N 27/26 response of this test Strip is essentially temperature inde
52 U.S. Cl. ................................. 204/403; 435/4; 435/14; pendent over relevant temperature ranges and is Substan
435/289.1 tially insensitive to the hematocrit of the patient.
58 Field of Search ..................................... 204/403, 415,
204/418; 435/287.1, 289.1, 817, 4, 14 2 Claims, 6 Drawing Sheets
U.S. Patent Sep. 14, 1999 Sheet 1 of 6 5,951,836

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U.S. Patent Sep. 14, 1999 Sheet 2 of 6 5,951,836

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U.S. Patent Sep. 14, 1999 Sheet 5 of 6 5,951,836

2O BLOOD AT 37°C

1O y = O.1OO9x + 4.7656
R2 = O.9945
O
O 1OO 2OO 3OO 4OO 5 OO
GLUCOSE mg/d
F. G. 6A

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CO
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BLOOD A 2CC

y = O.1O68x +5.1522
R2 = O.9867
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GLUCOSE mg/dl
F.G. 6B
U.S. Patent Sep. 14, 1999 Sheet 6 of 6 5,951,836

O 5 1 O 15 20 25 3O 35
TME (SECONDS)
F.G. 8A

25

15

1O

O 5 1 O 15 20 25 3O 35
TIME (SECONDS)
F.G. 8B
 5,951,836
1 2
DISPOSABLE GLUCOSE TEST STRP AND This fact is clearly demonstrated by the much lower currents
METHOD AND COMPOSITIONS FOR measured in blood Samples than in plasma Samples or other
MAKING SAME aqueous media, and can result in erroneous readings. A
further consequence is that the transients are often "lumpy'
This application is a continuation of U.S. Pat. application as the electrode breaks up in a chaotic manner. Lumpy
Ser. No. 08/601.223 filed Feb. 14, 1996, issue Jan. 13, 1998 transients either give rise to erroneous readings or rejected
U.S. Pat. No. 5,708,207, which is incorporated herein ref Strips, neither of which are acceptable.
CCCC. In addition to the potential for electrode breakup of
BACKGROUND OF THE INVENTION Screen-printed carbon-based electrodes, known electrodes
used in disposable glucose test Strips have been kinetically
This application relates to disposable glucose test Strips controlled, i.e., the current depends on the rate of conversion
for use in electrochemical determinations of blood glucose, of glucose by the enzyme. Because the response measured
and to methods and compositions for use in making Such by the instrument represents a balance between the reaction
Strips. of enzyme and mediator, enzyme and glucose and enzyme
15 and oxygen, and because each of these reactions has its own
Glucose monitoring is a fact of everyday life for diabetic
individuals, and the accuracy of Such monitoring can liter dependence on temperature, the response of a kinetically
ally mean the difference between life and death. To accom controlled test Strip is very Sensitive to the temperature of the
modate a normal life Style to the need for frequent moni Sample. Substantial variation in the measured glucose value
toring of glucose levels, a number of glucose meters are now can therefore occur as a result of variations in Sample
available which permit the individual to test the glucose handling.
level in a small amount of blood. Because of the importance of obtaining accurate glucose
Many of these meters detect glucose in a blood Sample readings to the well-being of a patient using the meter and
electrochemically, by detecting the oxidation of blood glu disposable test Strips, it would be highly desirable to have a
cose using an enzyme Such as glucose oxidase provided as 25
glucose test Strip which did not Suffer from these drawbacks,
part of a disposable, Single use electrode System. Examples and which therefore provided a more consistent and reliable
of devices of this type are disclosed in European Patent No. indication of actual blood glucose values, regardless of
0 127958, and U.S. Pat. Nos. 5,141,868, 5.286,362, 5,288, actual conditions. It is therefore an object of the present
636, and 5,437,999 which are incorporated herein by refer invention to provide disposable glucose test Strips which are
CCC. not prone to electrode breakup on contact with a Sample.
In general, existing glucose test Strips for use in electro It is a further object of this invention to provide glucose
chemical meters comprise a Substrate, working and refer test Strips which provide a glucose reading that is essentially
ence electrodes formed on the Surface of the Substrate, and independent of the hematocrit of the Sample.
a means for making connection between the electrodes and It is a further object of the present invention to provide
the meter. The working electrode is coated with an enzyme 35 glucose test Strips which are Substantially independent of
capable of oxidizing glucose, and a mediator compound temperature over a range between normal body temperature
which transferS electrons from the enzyme to the electrode and room temperature.
resulting in a measurable current when glucose is present. It is a further object of the invention to provide test strips
Representative mediator compounds include ferricyanide, which provide a Substantially flat current transient, without
metallocene compounds Such as ferrocene, quinones, phena 40 Significant decay for periods of at least 10 Seconds after the
Zinium salts, redox indicator DCPIP, and imidazole peak current level is obtained.
Substituted OSmium compounds.
Working electrodes of this type have been formulated in SUMMARY OF THE INVENTION
a number of ways. For example, mixtures of conductive The present invention provides an improved disposable
carbon, glucose oxidase and a mediator have been formu 45
glucose test Strip for use in a test meter of the type which
lated into a paste or ink and applied to a substrate. EP 0127 receives a disposable test Strip and a Sample of blood from
958 and U.S. Pat. No. 5.286,362. In the case of disposable a patient and performs an electrochemical analysis of the
glucose Strips, this application is done by Screen printing in amount of glucose in the Sample. The test Strip comprises:
order to obtain the thin layers suitable for a small flat test
Strip. The use of Screen printing, however, introduces prob 50 (a) a Substrate;
lems to the operation of the electrode. (b) a reference electrode,
Unlike a thicker carbon paste electrode which remains (c) a working electrode; and
fairly intact during the measurement, Screen printed elec (d) means for making an electrical connection
trodes formed from carbon pastes or inks are prone to break between the reference and working electrode and a glucose
up on contact with the Sample. The consequences of this 55 test meter. The working electrode comprises a conductive
breakup are two-fold. Firstly, the components of the elec base layer disposed on the Substrate and a non-conductive
trode formulation are released into Solution. Once these coating disposed over the conductive base layer. The non
components drift more than a diffusion length away from the conductive coating comprises a filler which has both hydro
underlying conductive layer, they no longer contribute phobic and hydrophilic Surface regions, an enzyme effective
toward the measurement, but in fact diminish the response 60 to oxidize glucose, e.g., glucose oxidase, and a mediator
by depleting inwardly-diffusing analyte. Secondly, the effective to transfer electrons from the enzyme to the con
breakup of the Screen printed electrode means that the ductive base layer. The filler is selected to have a balance of
effective electrode area is falling over time. hydrophobicity and hydrophilicity Such that on drying it
The combination of these two effects results in current forms a two-dimensional network on the Surface of the
transients which fall rapidly from an initial peak over the 65 conductive base layer. Preferred filler are non-conductive
period of the measurement, and a high Sensitivity to oxygen Silica fillers. The response of this test Strip is dependent on
which quickly competes with the mediator for the enzyme. the diffusion rate of glucose, not on the rate at which the
 5,951,836
3 4
enzyme can oxidize glucose, Such that the performance of makes contact with working electrode track 15, and is close
the test Strip is essentially temperature independent over too but not contacting the end of reference electrode track
relevant temperature ranges. Further, the Silica appears to 15.
form a two-dimensional network which excludes red blood The insulating layer 18 can be formed from polyester
cells, thus rendering the test Strip Substantially insensitive to based printable dielectric materials such as ERCON R488
the hematocrit of the patient. B(HV)-B2 Blue.
BRIEF DESCRIPTION OF THE DRAWINGS
The key to the performance achieved using the present
invention is in the nature of the coating 17. This coating
FIGS. 1A and 1B shows an electrode structure useful in contains a filler which has both hydrophobic and hydrophilic
a disposable test Strip in accordance with the invention; Surface regions, an enzyme which can oxidize glucose, and
FIG.2 shows a test strip in accordance with the invention; a mediator which can transfer electrons from the enzyme to
FIGS. 3A-3C show the current measured as a function of
the underlying conductive base layer 16.
glucose concentration for three different hematocrit levels, A preferred filler for use in the coating 17 is silica. Silica
is available in a variety of grades and with a variety of
FIG. 4 shows the relationship of the glucose 15
surface modifications. While all silica compounds tested
concentration dependence of the measured current as a resulted in a product which could measure glucose under
function of hematocrit; Some conditions, the Superior performance characteristics of
FIGS. 5A-5C show the current measured as a function of glucose test Strip of the invention are obtained when a Silica
glucose in blood and a control solution for three different having a Surface modification to render it partially hydro
variations of the conductive base layer; phobic is used. Materials of this type include Cab-O-Sil
FIG. 6A and 6B show the current measured as a function TS610, a silica which is modified by partial surface treat
of glucose at two different temperatures, ment with methyl dichlorosilane; Cab-o-Sil 530, a silica
FIG. 7 shows a further embodiment of a glucose test strip which is modified by full surface treatment with hexamethyl
according to the invention; and 25
disilaZane; Spherisorb C4 silica, which is surface modified
FIGS. 8A and 8B show a current transients observed using with 4 carbon chains, and other similarly modified by Silicas,
or combinations thereof.
a test Strip according to the invention and a commercial
carbon-based test Strip. Silica with a surface modification which is too hydropho
bic should be avoided, however, since it has been observed
DETAILED DESCRIPTION OF THE that C18-modified silica is too hydrophobic to form a
INVENTION printable ink.
During the process of manufacturing the ink of the
FIGS. 1A and 1B show electrodes useful in a disposable invention, the particles are broken down by homogenization
test strip in accordance with the invention. As shown, the to expose hydrophilic inner portions of the Silica particles.
electrodes are formed on a Substrate 10. On the Substrate 10
are placed a conductive base layer 16, a working electrode 35 The actual particles present in the ink therefore have both
hydrophilic and hydrophobic regions. The hydrophilic
track 14, a reference electrode track 15, and conductive regions form hydrogen bonds with each other and with
contacts 11, 12, and 13. An insulating mask 18 is then Water.
formed, leaving a portion of the conductive base layer 16, When this material is formulated into an ink as described
and the contacts 11, 12 and 13 exposed. A region of a 40 below in Example 1, and Screen printed onto the conductive
working coating 17 is then applied over the insulating mask base layer 16, the dual nature of the material causes it two
18 to make contact with conductive base layer 16. form layers of two-dimensional networks which take form
The assembly shown in FIG. 1 provides a fully functional as a kind of honeycomb. On rehydration, this layer does not
assembly for the measurement of blood glucose when con break up, but Swells forming a gelled reaction Zone in the
nected to a meter. Advantageously, however, the electrode 45 vicinity of the underlying conductive base layer 16. Enzyme,
Strips of the invention are finished by applying a polyester mediator and glucose move freely within this Zone, but
mesh 21 over the region of the working coating 17 of the interfering Species Such as red blood cells containing oxy
electrode assembly 22, and then a top cover 23 to prevent genated hemoglobin are excluded. This results in a device
splashing of the blood sample. (FIG. 2) The polyester mesh which the amount of current generated in response to a given
acts to guide the Sample to the reference electrode, thereby 50 amount of glucose varies by less than 10 percent over a
triggering the device and initiating the test. hematocrit range of 40 to 60%, and which is thus substan
The substrate 10 used in making the test strips of the tially insensitive to the hematocrit of the Sample, and in fact
invention can be any non-conducting, dimensionally stable performs Substantially the same in blood as in a cell-free
material Suitable for insertion into a glucose test meter. control solution. (FIGS. 3A-C, FIG. 4 and FIG. 5A-5C)
Suitable materials include polyester films, for example a 330 55 Furthermore, the gelled reaction Zone presents a greater
micron polyester film, and other insulating Substrate mate barrier to entry of glucose which makes the device diffusion,
rials. rather than kinetically limited. This leads to a device in
The working electrode track 15, the reference electrode which the measured current varies by less than 10percent
track 14, and conductive contacts 11, and 12 can be formed over a temperature range from 20° C. to 37 C. and which
from essentially any conductive material including Silver, 60 is thus essentially temperature independent. (FIGS. 6A and
Ag/AgCl, gold, or platinum/carbon. 6B)
The conductive base layer 16 is preferably formed from The working layer is advantageously formed from an
conductive carbon. Preferred conductive carbon are ERCON aqueous composition containing 2 to 10% by weight, pref
ERC1, ERCON ERC2 and Acheson Carbon Electrodag 423. erably 4 to 10% and more preferably about 4.5% of a binder
Carbon with these specifications is available from Ercon Inc. 65 such as hydroxyethylcellulose or mixtures of hydroxyeth
(Waltham, Mass., USA), or Acheson Colloids, (Princes ylcellulose with alginate or other thickeners; 3 to 10% by
Rock, Plymouth, England). The conductive base layer 16 weight, preferably 3 to 5% and more preferably about 4%
 5,951,836
S 6
silica; 8 to 20% by weight, preferably 14 to 18% and more R-414 (DPM-68)1.25 bioelectrode sensor coating material
preferably about 16% of a mediator such as ferricyanide; and dried. One Working track which makes contact with the
and 0.4 to 2% by weight, preferably 1 to 2% and more carbon pad and one reference track is printed for each carbon
preferably about 1.6% of an enzyme Such as glucose pad in the array.
oxidase, assuming a specific activity of about 250 units/mg, A dielectric layer is then printed using ERCON R488-B
or about 1000 to 5000 units per gram of ink formulation. (HV)-B2 Blue and dried. The dielectric layer is printed in a
The working layer may also include additional ingredients pattern which coverS Substantially all of each devices, leav
without departing from the Scope of the invention. For ing only the contacts, the tip of the reference electrode and
example, the nonconducting layer may include an antifoam. the carbon pads uncovered.
In addition, the nonconducting layer may be formulated with On top of the dielectric layer the ink of Example 1 is used
a buffering agent to control the pH of the reaction Zone. The to form a working layer overlaid on top of each conductive
pH may be maintained at a level within the range from about carbon pad.
pH 3 to pH10. It is of particular utility to maintain the pH Polyester mesh strips (Scrynel PET230 HC) are then laid
of the device at a level above 8 because at this pH oxygen down across the Substrate in lines, covering the reactions
bound to hemoglobin is not released. Further, at this pH, the 15
reaction rate of glucose oxidase with oxygen is very low. areas exposed by the windows in the dielectric. An 5 mm
Thus, Selection of an appropriate pH can further Stabilize the wide polyester Strip (50 microns thick) is then applied over
performance of the test Strip against the effects of varying the top of the mesh Strips, and the edges of the electrodes are
hematocrit. heat sealed. Finally, the substrate is cut up to provide 50
individual electrodes, for example having a Size of 5.5 mm
FIG. 7 shows an alternative embodiment of the invention. wide and 30 mm long.
In this embodiment, a Second working layer 71 is disposed
over the first working layer 17. This layer is formed from a EXAMPLE 3
composition which is identical to the first working layer Test Strips manufactured using the ink formulation of
except that the enzyme or both the enzyme and the mediator 25 Example 1 in the manner described in Example 2 were
are omitted. This layer further isolates the conductive base placed in a test meter with an applied voltage of 500 mV and
layer from contact with oxygen-carrying red blood cells, used to test blood Samples having varying glucose concen
thus reducing the effects of oxygen. Furthermore, to the trations and hematocrits ranging from 40% to 60%. FIGS.
extent that enzyme may tend to diffuse away from the 3A-3C show the current measured 25 seconds after applying
Surface of the electrode during the course of the the Voltage as a function of the glucose concentration, and
measurement, this layer provides an increased region in FIG. 4 plots the Slope of the glucose response as a function
which it will have mediator available for the transfer of
electrons. of hematocrit. AS can be seen, the indicators produce highly
reproducible current levels which are essentially indepen
EXAMPLE 1. dent of hematocrit.
35
A non-conducting formulation for preparation of the EXAMPLE 4
working layer 17 was made as follows. 100 ml of 20 mM
aqueous trisodium citrate was adjusted to pH 6 by the Glucose test Strips in accordance with the invention were
addition of 0.1 M citric acid. To this 6 g of hydroxyethyl made in accordance with Example 2, except the non
cellulose (HEC) was added and mixed by homogenization. 40
conductive layer was formed with 7 g Spherisorb C4 and 1
The mixture was allowed to stand overnight to allow air g Cab-o-Sil TS610. This formulation was laid down on two
bubbles to disperse and then used as a Stock Solution for the three different types of carbon-containing conductive base
formulation of the coating composition. layerS as follows:
2 grams Cab-o-Sil TS610 silica and 0.1 grams of Dow A: Ercon EC1
Corning antifoam compound was gradually added by hand 45 B: Ercon EC2
to 50 grams of the HEC solution until about Ysths of the total C: Ercon EC2 on top of Acheson Carbon, Electrodag 423
amount has been added. The remainder is added with mixing SS.
by homogenization. The mixture is then cooled for ten These test Strips were used to measure varying levels of
minutes in a refrigerator. 8 g of potassium hexacyanoferrate glucose in either a control solution (One Touch Control
(III) is then added and mixed until completely dissolved. 50 Solution, LifeScan Inc.) containing glucose in an inert Solu
Finally, 0.8 g of glucose oxidase enzyme preparation (250 tion or in blood at an applied voltage of 425 mV. The current
Units/mg) is added and the thoroughly mixed into the observed 25 Seconds after the Voltage was applied was
Solution. The resulting formulation is ready for printing, or measured. FIGS. 5A-5C show the results obtained for the
can be Stored with refrigeration. three formulations, A, B, and C, respectively. In all cases, the
EXAMPLE 2 55 Slope of the line showing the response of the meter to
different glucose concentrations was essentially the same
To prepare glucose test Strips using the ink formulation of whether the measurement were made in blood or the control
Example 1, a Series of patterns are used to Screen print layers Solution. Thus, this further demonstrates the independence
onto a 330 micron polyester substrate (Melinex 329). The of the test Strips of the invention from the oxygen content
first step is the printing of carbon pads. An array of 10x50 60 and hematocrit of the Sample, as well as the ability to use
pads of carbon is formed on the 103, Surface of the polyester varied material as the conductive base layer.
substrate by printing with EC2 carbon. (Ercon) The printed EXAMPLE 5
Substrate is then passed through a heated dryer, and option
ally cured at elevated temperature (e.g. 70° C.) for a period Test Strips prepared in accordance with Example 2were
of 1 to 3 weeks. 65 tested at two different sample temperatures, namely 37 C.
Next, an array of Silver/silver chloride connecting tracks and 20° C. using an applied voltage of 425 mV. FIGS. 6A
and contacts is printed onto the substrate using ERCON and 6B show the current measured 25 Seconds after applying
 5,951,836
7 8
the Voltage as a function of glucose concentration. AS can be the initial response from the test Strip. This makes timing of
Seen, the Slopes of the two lines are essentially identical the measurement difficult if peak current values are to be
(0.1068 at 20° C. versus 0.1009 at 37° C), thus demonstrat captured, or reduces the dynamic range of the meter is
ing that the test Strips provide essentially temperature currents must be measrured after Substantial decay has
independent behavior over a temperature range from ambi occurred. Thus, the test Strips of the invention are advanta
ent to physiological temperatures. geous in that they provide test Strips in which the amount of
current generated in response to a given amount of glucose
EXAMPLE 6 decays by less than 50% in the 5 seconds following peak
The current transient was measured for a test Strip pre current generation.
pared in accordance with Example 2 and for a commercial We claim:
test Strip made with a carbon-containing ink. The results are 1. An aqueous composition comprising a binder, a filler
shown in FIGS. 8A and 8B. As shown, the test strip of the having both hydrophobic and hydrophilic Surface regions, at
invention (FIG. 8A) provides a very flat transient which least one of an enzyme effective to oxidize glucose and an
maintains more than 50% of the peak current for a period of electron transfer mediator.
15 2. The composition of claim 1, wherein the filler is
more than 25 Seconds after the initial response from the test non-conductive.
Strip. In contrast, the carbon-based electrode exhibited an
almost immediate decay in the current, having lost 50% of
the peak current in a period of the first 1 to 2 Seconds after
 US005951836C1

(12) EX PARTE REEXAMINATION CERTIFICATE (9960th)

United States Patent (10) Number: US 5,951,836 C1
McAleer et al. (45) Certificate Issued: *Nov. 26, 2013

(54) DISPOSABLE GLUCOSE TEST STRIP AND (52) U.S. Cl.

METHOD AND COMPOSITIONS FOR USPC .............. 205/777.5: 204/403.04; 204/403.11;
MAKING SAME 435/14; 435/289.1; 435/4
(58) Field of Classification Search
(75) Inventors: Jerome F. McAleer, Wantage (GB); None
David Scott, Witney (GB); Geoff Hall, See application file for complete search history.
Inverness (GB); Manuel Alvarez-Icaza,
Inverness (GB); Elliot V. Plotkin, (56) References Cited
Inverness (GB) To view the complete listing of prior art documents cited
(73) Assignee: Lifescan Scotland Limited, Inverness during the proceeding for Reexamination Control Number
(GB) 90/012,536, please refer to the USPTO's public Patent
Application Information Retrieval (PAIR) system under the
Reexamination Request: Display References tab.
No. 90/012,536, Sep. 13, 2012 Primary Examiner — Krisanne Jastrzab
Reexamination Certificate for:
Patent No.: 5,951,836
(57) ABSTRACT
Issued: Sep. 14, 1999 An improved disposable glucose test strip for use in a test
Appl. No.: 09/005,710 meter of the type which receives a disposable test strip and a
Filed: Jan. 12, 1998 sample of blood from a patient and performs an electrochemi
cal analysis is made using a working formulation containing
(*) Notice: This patent is subject to a terminal dis a filler, an enzyme effective to oxidize glucose, e.g., glucose
claimer. oxidase, and a mediator effective to transfer electrons from
the enzyme. The working formulation is printed over a con
Related U.S. Application Data ductive carbon base layer to form a working electrode. The
filler, for example a silica filler, is selected to have a balance
(63) Continuation of application No. 08/601.223, filed on of hydrophobicity and hydrophilicity such that one drying it
Feb. 14, 1996, now Pat. No. 5,708,247. forms a two-dimensional network on the surface of the con
ductive base layer. The response of this test strip is essentially
(51) Int. Cl. temperature independent over relevant temperature ranges
CI2O I/00 (2006.01) and is substantially insensitive to the hematocrit of the
GOIN33/50 (2006.01) patient.
 US 5,951,836 C1
1.
EX PARTE
REEXAMINATION CERTIFICATE
ISSUED UNDER 35 U.S.C. 307
THE PATENT IS HEREBY AMENDED AS
INDICATED BELOW.

Matter enclosed in heavy brackets appeared in the

patent, but has been deleted and is no longer a part of the 10
patent; matter printed in italics indicates additions made
to the patent.
AS A RESULT OF REEXAMINATION, IT HAS BEEN
DETERMINED THAT: 15

Claim 2 is cancelled.
Claim 1 is determined to be patentable as amended.
1. An aqueous composition comprising a binder, a filler
having both hydrophobic and hydrophilic surface regions, at 20
least one of an enzyme effective to oxidize glucose, and an
electron transfer mediator, wherein the composition is non
conductive.