(12)
`
`United States Patent
`McAleer et al.
`
`(10) Patent N0.:
`(45) Date of Patent:
`
`US 6,241,862 B1
`Jun. 5, 2001
`
`US006241862B1
`
`(54) DISPOSABLE TEST STRIPS WITH
`
`INTEGRATED REAGENT/BLOOD
`
`SEPARATION LAYER
`
`(75) Inventorsl J Home E McAleeB Wantage; Manuel
`Alvarez-Icaza, Inverness; Geo?' Hall,
`Inverness; Elliot V. Plotkin, Inverness;
`David Scott,
`OXOH; Oliver W
`H. Davies, Inverness, all of (GB)
`
`(73) Assignee: Inverness Medical Technology, Inc.,
`Waltham, MA (US)
`
`( * ) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`
`_
`(21) Appl' NO" 09/228’855
`(22) Filed;
`Jam 12, 1999
`
`Related US. Application Data
`
`(63) Continuation-in-part of application No. 09/005,710, ?led on
`Jan. 12, 1998, now Pat. No. 5,951,836, which is a continu-
`ation Of application N0. 08/601,223, ?led On Feb. 14, 1996,
`now Pat. No. 5,708,247.
`7
`........ .. G01N 27/26
`(51) Int. Cl. ........................................
`(52) U..S. Cl. ........................ .. 204/403, 204/415, 427/213
`Fleld of Search ................................... ..
`205/777-5, 778; 427/211, 212, 2-13
`
`(56)
`
`References Cited
`
`4,195,129
`4,418,148
`4,655,880
`4,876,205
`4,966,671
`5’12O’42O
`
`U'S' PATENT DOCUMENTS
`3/1980 Fukui et a1. ....................... .. 435/182
`11/1983 Oberhardt ,,
`435/179
`4/1987 Liu ........... ..
`204/1 T
`10/1989 Green eta1~
`436/66
`10/1990 Nylancler ct a1~
`~~ 2O4/153-14
`2/
`ganllilal etlal' '
`""" "
`M103
`51992 czlrltereett i1"
`435/288
`8/1992 Shanks e161. .
`5,141,868
`435/174
`5,185,256 * 2/1993 Nankai et al. .
`5,262,035
`11/1993 Gregg et a1. ....................... .. 204/403
`
`11/1993 McAleer et a1. ................... .. 204/403
`
`5,264,106
`
`253338
`
`511333 13°11“ “$11
`
`3321333
`
`
`
`
`
`, , 5,378,628
`
`
`
`0 mane a. . 1/1995 Griitzel et a1. . . . . . . .
`
`. . . .. 435/288
`
`435288
`..
`l.
`8 1995 D' b ld t
`5437 999
`5’601’694 * 2/1997 8466163616615 ....................... .. 204/415
`7
`7
`
`FOREIGN PATENT DOCUMENTS
`
`170375 * 5/1986 (EP)
`207370 A2 * 11/1994 (EP)
`9702487 * 5/1986 (W0)
`89/08713
`9/1989 (WO)
`9427140 A1 * 11/1994 (WO)
`9427140
`11/1994 (W0)
`OTHER PUBLICATIONS
`
`abstract of JP55124060, Mar. 1979.*
`English language translation of JP5510583, Jan. 1980.*
`English language translation of JP5510584, Jan. 1980.*
`English language translation of JP55—10581, Jan. 1980*
`
`*
`
`.
`
`.
`
`cued by exammer
`Primary Examiner—Jill Warden
`Assistant Examiner—A1eX Noguerola
`'
`(74) Attorney’ Agent’ Or Flrm—oppedahl & Larson LLP
`(57)
`ABSTRACT
`
`An improved disposable glucose test Strip for use in a test
`meter of the type which receives a disposable test Strip and
`a Sample of blood from a patient and performs an electro
`chemical analysis is made using a non-conductive integrated
`reagent/blood separation layer containing a ?ller, an enZyme
`effective to oxidize glucose, e.g., glucose oxidase, and a
`mediator effective to transfer electrons from the enZyme.
`The integrated layer formulation is printed over a conductive
`Carbon element to form a Working 919990919- The ?ller, for
`example a silica ?ller, is selected to have a balance of
`hydrophobicity and hydrophilicity such that one drying it
`forms a two-dimensional network on the surface of the
`conductive element. The response of this test strip is essen
`tially temperature independent over relevant temperature
`r?nges 'and is substantially insensitive to the hematocrit of
`t e Panam
`
`34 Claims, 9 Drawing Sheets
`
`2
`
`1 Z \
`
`///l
`
`1O
`
`Case 3:14-cv-00274-RJC-DSC Document 6-1 Filed 07/21/14 Page 1 of 19
`
`

`
`U.S. Patent
`
`Jun. 5, 2001
`
`Sheet 1 0f 9
`
`US 6,241,862 B1
`
`FIG. 1A
`
`16
`
`1? 14'
`
`3Q
`
`1e
`
`/
`
`12
`
`/
`
`FIG.1B
`
`Case 3:14-cv-00274-RJC-DSC Document 6-1 Filed 07/21/14 Page 2 of 19
`
`

`
`U.S. Patent
`
`Jun. 5, 2001
`
`Sheet 2 0f 9
`
`US 6,241,862 B1
`
`FIG. 2
`
`
`
`SLOPE FROM FIGS 3A~3C
`
`
`
`
`
`4O
`
`45
`
`55
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`HEIVIATOCRIT (0/0)
`FIG. 4
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`16
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`)2
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`
`'10
`
`Case 3:14-cv-00274-RJC-DSC Document 6-1 Filed 07/21/14 Page 3 of 19
`
`

`
`U.S. Patent
`
`Jun. 5, 2001
`
`Sheet 3 0f 9
`
`US 6,241,862 B1
`
`70
`6O_
`5O _
`
`y=0.096x+10.477
`R2 =O.9681
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`200
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`FIG. 3A
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`
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`
`I
`
`500
`
`Case 3:14-cv-00274-RJC-DSC Document 6-1 Filed 07/21/14 Page 4 of 19
`
`

`
`U.S. Patent
`
`Jun. 5, 2001
`
`Sheet 4 of 9
`
`US 6,241,862 B1
`
`1OO
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`
`Case 3:14-cv-00274-RJC-DSC Document 6-1 Filed 07/21/14 Page 5 of 19
`Case 3:14—Cv—OO274—RJC—DSC Document 6-1 Filed 07/21/14 Page 5 Of 19
`
`

`
`U.S. Patent
`
`Jun. 5, 2001
`
`Sheet 5 0f 9
`
`US 6,241,862 B1
`
`60
`
`I
`
`I
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`
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`
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`
`RESPONSE
`
`BLOOD AT 37°C
`
`y= 0.1009x +4.7656
`R2 = 0.9945
`
`_
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`
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`
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`200
`GLUCOSE mg/dl
`
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`
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`
`FIG. 6A
`
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`
`,
`
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`
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`
`RESPONSE
`
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`
`40 -
`
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`
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`
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`
`FIG. 6B
`
`Case 3:14-cv-00274-RJC-DSC Document 6-1 Filed 07/21/14 Page 6 of 19
`
`

`
`U.S. Patent
`
`Jun. 5, 2001
`
`Sheet 6 6f 9
`
`US 6,241,862 B1
`
`O 5 1O 15 2O 25 5O 55
`TIME (SECONDS)
`FIG. 8A
`
`5 2
`
`2 All 4‘
`
`_ _ _
`
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`
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`
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`
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`5
`TIME (SECONDS)
`
`FIG. 8B
`
`Case 3:14-cv-00274-RJC-DSC Document 6-1 Filed 07/21/14 Page 7 of 19
`
`

`
`U.S. Patent
`
`Jun. 5, 2001
`
`Sheet 7 0f 9
`
`US 6,241,862 B1
`
`FIG. 9A
`
`FIG. 9C
`
`Case 3:14-cv-00274-RJC-DSC Document 6-1 Filed 07/21/14 Page 8 of 19
`
`

`
`U.S. Patent
`
`Jun. 5, 2001
`
`Sheet 8 of9
`
`US 6,241,862 B1
`
`H
`
`F L/103 U H
`H II
`Li 'J|J1\\1
`
`y/1o1
`
`100/’
`
`FIG. 10A
`
`II
`
`.
`
`FIG 10s
`
`II
`
`1'11: {H {in M‘
`I l: h
`W
`
`“In
`
`FIG. 10C
`
`FIG. 10D
`
`Case 3:14-cv-00274-RJC-DSC Document 6-1 Filed 07/21/14 Page 9 of 19
`
`

`
`U.S. Patent
`
`Jun. 5, 2001
`
`Sheet 9 0f 9
`
`US 6,241,862 B1
`
`
`ETIJL IWMU 1.
`rllllullllllh ‘IIPIIIHHIIWIIIWIHM H U CV
`
`
`
`FIG. 10E
`
`FIG.1OG
`
`Case 3:14-cv-00274-RJC-DSC Document 6-1 Filed 07/21/14 Page 10 of 19
`
`

`
`US 6,241,862 B1
`
`1
`DISPOSABLE TEST STRIPS WITH
`INTEGRATED REAGENT/BLOOD
`SEPARATION LAYER
`
`This application is a continuation-in-part of US. patent
`application Ser. No. 09/005,710, ?led Jan. 12, 1998, now
`US. Pat. No. 5,951,836, Which is a continuation of US.
`patent application Ser. No. 08/601,223, ?led Feb. 14, 1996,
`issued Jan. 13, 1998 as US. Pat. No. 5,708,247, Which are
`incorporated herein by reference.
`
`BACKGROUND OF THE INVENTION
`
`This application relates to disposable test strips for use in
`electrochemical determinations of blood analytes such as
`glucose, and to methods and compositions for use in making
`such strips.
`Glucose monitoring is a fact of everyday life for diabetic
`individuals, and the accuracy of such monitoring can liter
`ally mean the difference betWeen life and death. To accom
`modate a normal life style to the need for frequent moni
`toring of glucose levels, a number of glucose meters are noW
`available Which permit the individual to test the glucose
`level in a small amount of blood.
`Many of these meters detect glucose in a blood sample
`electrochemically, by detecting the oxidation of blood glu
`cose using an enZyme such as glucose oxidase provided as
`part of a disposable, single-use electrode system. Examples
`of devices of this type are disclosed in European Patent No.
`0 127 958, and US. Pat. Nos. 5,141,868, 5,286,362, 5,288,
`636, and 5,437,999 Which are incorporated herein by refer
`ence.
`In general, existing glucose test strips for use in electro
`chemical meters comprise a substrate, Working and refer
`ence electrodes formed on the surface of the substrate, and
`a means for making connection betWeen the electrodes and
`the meter. The Working electrode is coated With an enZyme
`capable of oxidiZing glucose, and a mediator compound
`Which transfers electrons from the enZyme to the electrode
`resulting in a measurable current When glucose is present.
`Representative mediator compounds include ferricyanide,
`metallocene compounds such as ferrocene, quinones, phena
`Zinium salts, redox indicator DCPIP, and imidaZole
`substituted osmium compounds.
`Working electrodes of this type have been formulated in
`a number of Ways. For example, mixtures of conductive
`carbon, glucose oxidase and a mediator have been formu
`lated into a paste or ink and applied to a substrate. EP 0 127
`958 and US. Pat. No. 5,286,362. In the case of disposable
`glucose strips, this application is done by screen printing in
`order to obtain the thin layers suitable for a small ?at test
`strip. The use of screen printing, hoWever, introduces prob
`lems to the operation of the electrode.
`Unlike a thicker carbon paste electrode Which remains
`fairly intact during the measurement, screen printed elec
`trodes formed from carbon pastes or inks are prone to break
`up on contact With the sample. The consequences of this
`breakup are tWo-fold. Firstly, the components of the elec
`trode formulation are released into solution. Once these
`components drift more than a diffusion length aWay from the
`underlying conductive layer, they no longer contribute
`toWard the measurement, but in fact diminish the response
`by depleting inWardly-diffusing analyte. Secondly, the
`breakup of the screen printed electrode means that the
`effective electrode area is falling over time.
`The combination of these tWo effects results in current
`transients Which fall rapidly from an initial peak over the
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`2
`period of the measurement, and a high sensitivity to oxygen
`Which quickly competes With the mediator for the enZyme.
`This fact is clearly demonstrated by the much loWer currents
`measured in blood samples than in plasma samples or other
`aqueous media, and can result in erroneous readings. A
`further consequence is that the transients are often “lumpy”
`as the electrode breaks up in a chaotic manner. Lumpy
`transients either give rise to erroneous readings or rejected
`strips, neither of Which are acceptable.
`In addition to the potential for electrode breakup of
`screen-printed carbon-based electrodes, knoWn electrodes
`used in disposable glucose test strips have been kinetically
`controlled, i.e., the current depends on the rate of conversion
`of glucose by the enZyme. Because the response measured
`by the instrument represents a balance betWeen the reactions
`of enZyme and mediator, enZyme and glucose and enZyme
`and oxygen, and because each of these reactions has its oWn
`dependence on temperature, the response of a kinetically
`controlled test strip is very sensitive to the temperature of the
`sample. Substantial variation in the measured glucose value
`can therefore occur as a result of variations in sample
`handling.
`A further challenge facing sensors for electrochemical
`glucose detection arises as a result of interference from
`blood cells present in the sample. The level of red blood cells
`is re?ected in the hematocrit reading. Typically, high hema
`tocrit samples results in readings that are loWer than the true
`value, While loW hematocrit samples result in readings that
`are higher because the blood cells tend to foul the surface of
`the electrode and limit electron transfer. Also, oxygen bound
`to the hemoglobin of red blood cells competes With the
`mediator for the reduced enZyme, thereby further diminish
`ing the glucose response. Attempts have been made to limit
`the hematocrit effect by adding a separately-deposited mem
`brane formed from a Water-insoluble hydrophobic polymer
`and a Water-soluble hydrophilic polymer in combination
`With a Water-soluble erythrocyte aggregating agent. to ?lter
`out blood components (see, US. Pat. No. 5,658,444, Which
`is incorporated herein by reference), but this adds an extra
`step to the manufacturing process, With associated increase
`in cost and often degraded performance in other areas such
`as precision.
`Because of the importance of obtaining accurate glucose
`readings to the Well-being of a patient using the meter and
`disposable test strips, it Would be highly desirable to have a
`glucose test strip Which did not suffer from these draWbacks,
`and Which therefore provided a more consistent and reliable
`indication of actual blood glucose values, regardless of
`actual conditions. It is therefore an object of the present
`invention to provide disposable glucose test strips Which
`provide a glucose reading that is essentially independent of
`the hematocrit of the sample, and Which include an inte
`grated reagent/blood separation layer.
`It is a further object of the present invention to provide an
`improved method for making disposable glucose test strips.
`SUMMARY OF THE INVENTION
`The present invention provides an improved disposable
`test strip for use in a test meter of the type Which receives
`a disposable test strip and a sample of blood from a patient
`and performs an electrochemical analysis of the amount of
`a blood analyte such as glucose in the sample. The test strip
`comprises:
`(a) a substrate;
`(b) a ?rst conductive element disposed on the substrate;
`(c) a second conductive element disposed on the substrate
`in sufficient proximity to the ?rst conductive element to
`
`Case 3:14-cv-00274-RJC-DSC Document 6-1 Filed 07/21/14 Page 11 of 19
`
`

`
`US 6,241,862 B1
`
`3
`allow the completion of an electrical circuit between
`the ?rst and second conductive elements When a sample
`of blood is placed on the test strip;
`(d) a non-conductive integrated reagent/blood separation
`layer disposed over the ?rst conductive element; and
`(e) contacts for making an electrical connection betWeen
`the ?rst and second conductive elements and the test
`meter. The integrated reagent/blood separation layer
`comprises reagents for the electrochemical detection of
`the analyte dispersed in a non-conductive matriX effec
`tive to eXclude blood cells from the surface of the ?rst
`conductive element While permitting access to the ?rst
`conductive element by soluble electroactive species. In
`one embodiment of the invention, a glucose test strip is
`formed With an integrated reagent/blood separation
`layer comprising a ?ller Which has both hydrophobic
`and hydrophilic surface regions, an enZyme effective to
`oXidiZe glucose, e.g., glucose oXidase, and a mediator
`effective to transfer electrons from the enZyme to the
`conductive element. The ?ller is selected to have a
`balance of hydrophobicity and hydrophilicity such that
`on drying the integrated reagent/blood separation layer
`forms a tWo-dimensional netWork on the surface of the
`conductive element. Preferred integrated reagent/blood
`separation layers comprise non-conductive silica ?llers
`in combination With materials such as hydroXyethyl
`cellulose
`The silica and HEC form a tWo
`dimensional netWork Which eXcludes red blood cells,
`thus rendering the test strip substantially insensitive to
`the hematocrit of the patient.
`In a preferred embodiment of the invention, the test strips
`are prepared With an insulation layer disposed over at least
`the ?rst conductive element. This insulation layer has an
`aperture formed in it Which is aligned With a portion of the
`?rst conductive element, and the integrated reagent/blood
`separation layer is disposed to make contact With the ?rst
`conductive element through this aperture.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIGS. 1A and 1B shoW an electrode structure useful in a
`disposable test strip in accordance With the invention;
`FIG. 2 shoWs a test strip in accordance With the invention;
`FIGS. 3A—3C shoW the current measured as a function of
`glucose concentration for three different hematocrit levels;
`FIG. 4 shoWs the relationship of the glucose
`concentration dependence of the measured current as a
`function of hematocrit;
`FIGS. 5A—5C shoW the current measured as a function of
`glucose in blood and a control solution for three different
`conductive elements;
`FIGS. 6A and 6B shoW the current measured as a function
`of glucose at tWo different temperatures;
`FIG. 7 shoWs a further embodiment of a glucose test strip
`according to the invention;
`FIGS. 8A and 8B shoW current transients observed using
`a test strip according to the invention and a commercial
`carbon-based test strip;
`FIGS. 9A—C shoW a three-step process for manufacture of
`test strips in accordance With the invention; and
`FIGS. 10A—10G shoW the manufacture of a test strip in
`accordance With the invention.
`
`10
`
`15
`
`25
`
`35
`
`45
`
`55
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`FIGS. 1A and 1B shoW electrodes useful in a disposable
`test strip in accordance With the invention. As shoWn, the
`
`65
`
`4
`electrodes are formed on a substrate 10. On the substrate 10
`are placed tWo conductive elements 14‘ and 16, connected by
`leads 14 and 15 to conductive contacts 11, 12, and 13. An
`insulating mask 18 is then formed, leaving at least a portion
`of conductive elements 14‘ and 16, and the contacts 11, 12
`and 13 eXposed. A non-conductive integrated reagent/blood
`separation layer 17 is then applied over the insulating mask
`18 to make contact With conductive element 16.
`The assembly shoWn in FIG. 1 provides a fully functional
`assembly for the measurement of a blood analyte When
`connected to a meter. Advantageously, hoWever, the elec
`trode strips of the invention are ?nished by applying a nylon
`or polyester mesh 21 over the sample application region
`de?ned by the location of the integrated reagent/blood
`separation layer 17 of the electrode assembly 22, and then a
`top cover 23 to prevent splashing of the blood sample. (FIG.
`2) The polyester mesh acts to guide the sample to the
`reference electrode, conductive element 14‘, thereby trigger
`ing the device and initiating the test.
`The utiliZation of a non-conductive integrated reagent/
`blood separation layer provides an important distinction
`from and advantage over knoWn test strips Which utiliZe a
`conductive reagent-containing slurry to print the reagents. In
`these systems, the printed slurry becomes a functional part
`of the electrode and charge transfer can take place at the
`outer surface of the reagent layer. If the layer is in direct
`contact With blood, i.e., When no intervening separation
`layer has been deposited, red and White blood cells, fat and
`proteins present in the sample can interact With the reagent
`layer and interfere With the measurement of the amount of
`analyte in the sample.
`In contrast, in the present invention, the integrated
`reagent/blood separation layer is non-conductive, and thus is
`not a part of the electrode either structurally or functionally.
`Charge transfer does not occur unless electroactive species
`pass through the openings/pores of the integrated reagent/
`blood separation layer to reach the underlying conductive
`element. Thus, the integrated reagent/blood separation layer
`provides a barrier to the passage of interferents such as cells
`and macromolecules to the conductive element resulting in
`a device With superior properties that is simpler to make.
`In achieving this result, it is particularly desirable that the
`integrated reagent/blood separation layer be deposited in
`such a Way that no portion of the conductive element 16 be
`directly eXposed to the sample When it is placed in the
`sample application region. The methodology described
`above, in Which an insulating layer With apertures providing
`access to the conductive elements 14‘ and 16 is utiliZed is
`particularly suited for achieving this result. Thus, as shoWn
`in FIGS. 9A—C, this methodology alloWs the formation of
`the test strip in only three steps. In the ?rst step (FIG. 9A),
`tWo conductive elements 14‘ and 16 and associated leads and
`contacts are deposited on a substrate. In a second step (FIG.
`9B), a layer of insulating material is deposited over the
`conductive elements. The insulating material has tWo aper
`tures 94 and 96, one in alignment With each of the conduc
`tive elements 14‘ and 16. In the third step, (FIG. 9C), the
`integrated reagent/blood separation layer 17 is deposited
`over the aperture 96. By making the deposited layer 17
`larger in dimensions than the aperture 96, the reagent layer
`completely covers the underlying conductive element such
`that it is not eXposed directly to the sample, thereby pro
`viding effective blood separation.
`The complete coverage of conductive element 16 also
`addresses another source of error Which can occur as a result
`of electrochemical oxidation or small molecules such as
`
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`US 6,241,862 B1
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`5
`ascorbic acid, uric acid and acetaminophen Which may be
`present in the sample. When present, the oxidation of these
`molecules at the surface of the electrode leads to spuriously
`elevated current levels, and thus an inaccurate measurement
`of the desired analyte, e.g. glucose. The integrated reagent/
`blood separation layer of invention Will not generally
`exclude these molecules, since they are small compared to
`the pore siZes observed. HoWever, by including a pH buffer
`in the integrated reagent/blood separation layer one can shift
`the local pH at the electrode surface to a level Where
`electrochemical potential of these species is higher. Thus,
`for example, the use of an integrated reagent/blood separa
`tion layer in Which the pH is buffered to a level of around pH
`5 Will substantially reduce the impact of these interferents.
`To maximiZe the effectiveness of this buffering, hoWever,
`the entire conductive element must be covered, since even a
`relatively small region of exposed (not buffered) electrode
`surface can result in a large interference current.
`Not only do the test strips of the invention provide
`performance bene?ts resulting from the separation of the
`conductive element from the blood sample, the test strips of
`the invention are also resistant to other sources of error. For
`example, during the period of a test, reagents may diffuse
`laterally aWay from the original deposit. If the reagent layer
`is deposited directly on the conductive element, these
`reagents Will continue to contribute to the measured signal.
`Any variations in convective diffusion from test to test (for
`example as a result of differences in temperature or differ
`ences in the handling of the instrument) Will therefore be
`manifested as irreproducibility in the signal. If the reagent
`layer overlaps the insulation print, hoWever, lateral diffusion
`aWay from the aperture Will not contribute to the signal and
`therefore Will not give rise to variations in the signal.
`In addition to providing a test strip With bene?cial
`properties, the methodology outlined in FIGS. 9A—C offers
`several advantages from a manufacturing perspective. First,
`if the reagent layer is printed directly onto the conductive
`element, the “active area” is de?ned by the area of the
`reagent layer. The precision of the test is therefore deter
`mined by the precision With Which the reagent layer can be
`printed. In contrast, by ?rst depositing an apertured insula
`tion layer de?ning the region of the contact betWeen the
`reagent layer and the underlying conductive element, the
`active area is de?ned by the siZe of the aperture in the
`insulation layer. Since insulation layers are typically printed
`using a ?ner screen, much better edge de?nition, and thus
`greater device precision can be achieved. Thus, neither the
`area of conductive element 16 nor of the integrated reagent/
`blood separation layer are critical to the performance char
`acteristics of the ?nished test strip. The conductive elements
`and the integrated reagent/blood separation layer may there
`fore be applied using techniques Which provide less preci
`sion than can be employed in other processes.
`It Will be appreciated by persons skilled in the art that,
`While both conductive elements must be accessible to elec
`troactive species in a sample disposed in the sample appli
`cation region, the important function of the insulation mask
`is to provide an aperture de?ning the contact region betWeen
`conductive element 16 and the integrated reagent/blood
`separation layer 17. Thus, in the limiting case, it is only
`necessary to form one aperture in the insulation layer. The
`second conductive element can be exposed along an edge of
`the insulation layer, or may be located on a facing surface in
`a folded electrode structure.
`The substrate 10 used in making the test strips of the
`invention can be any non-conducting, dimensionally stable
`material suitable for insertion into a glucose test meter.
`
`10
`
`15
`
`25
`
`35
`
`45
`
`55
`
`65
`
`6
`Suitable materials include polyester ?lms, for example a 330
`micron polyester ?lm, and other insulating substrate mate
`rials such as polyvinyl chloride (PVC) and polycarbonate.
`The conductive elements and associated leads and con
`tacts can be formed from essentially any conductive material
`including silver, Ag/AgCl, gold, or platinum/carbon, and
`need not all be formed from the same material. The con
`ductive element 16 is preferably formed from conductive
`carbon. Preferred conductive carbon are ERCON ERCl,
`ERCON ERC2 and Acheson Carbon Electrodag 423. Car
`bon With these speci?cations is available from Ercon Inc.
`(Waltham, Mass., USA), or Acheson Colloids, (Princes
`Rock, Plymouth, England). The conductive element 16
`makes contact With Working electrode track 15, and is close
`to, but not contacting conductive element 14‘ disposed as the
`end of reference electrode track 14.
`The insulating layer 18 can be formed from polyester
`based printable dielectric materials such as ERCON R488
`B(HV)-B2 Blue. The top cover 23 is suitably formed from
`a polyester strip or a “hot melt” coated plastic.
`The test strips of the present invention do not require the
`formation of a discrete exit port to permit air to escape from
`the device as sample enters the electrode chamber but
`instead uses a distributed exit along all of the edges of the
`mesh. As the sample ?uid Wicks along the mesh, air seeps
`out of the edges of the mesh all around the device under
`neath the top layer. The sample ?uid does not seep out
`because the insulation layer imparts signi?cant hydropho
`bicity to that part of the mesh. The liquid sample therefore
`remains in the central hydrophilic region.
`The key to the performance achieved using the present
`invention is in the nature of the integrated reagent/blood
`separation layer 17. This layer can be formed from a mixture
`containing a ?ller Which has both hydrophobic and hydro
`philic surface regions, and in the case of a glucose test strip,
`an enZyme Which can oxidiZe glucose, and a mediator Which
`can transfer electrons from the enZyme to the underlying
`conductive element layer 16. This layer is suitably formed
`by formulating an ink Which contains the ?ller, the enZyme
`and the mediator in a suitable carrier and using this ink to
`print the layer 17 onto the device.
`A preferred ?ller for use in the layer 17 is silica. Silica is
`available in a variety of grades and With a variety of surface
`modi?cations. While all silica compounds tested resulted in
`a product Which could measure glucose under some
`conditions, the superior performance characteristics of glu
`cose test strip of the invention are obtained When a silica
`having a surface modi?cation to render it partially hydro
`phobic is used. Materials of this type include Cab-O-Sil
`TS610, a silica Which is modi?ed by partial surface treat
`ment With methyl dichlorosilane; Cab-o-Sil 530, a silica
`Which is modi?ed by full surface treatment With hexamethyl
`disilaZane; Spherisorb C4 silica, Which is surface modi?ed
`With 4 carbon chains; and other similarly modi?ed silicas, or
`combinations thereof. Silica With a surface modi?cation
`Which is too hydrophobic should be avoided. For example,
`it has been observed that C18-modi?ed silica is too hydro
`phobic to form a printable ink.
`During the process of manufacturing the ink of the
`invention, the particles are broken doWn by homogeniZation
`to expose hydrophilic inner portions of the silica particles.
`The actual particles present in the ink therefore have both
`hydrophilic and hydrophobic regions. The hydrophilic
`regions form hydrogen bonds With each other and With
`Water.
`When this material is formulated into an ink as described
`beloW in Example 1, and screen printed onto the conductive
`
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`

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`US 6,241,862 B1
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`7
`element 16, the dual nature of the material causes it tWo form
`layers of tWo-dimensional networks Which take form as a
`kind of honeycomb Which is visible upon microscopic
`examination. On rehydration, this layer does not break up,
`but sWells to form a gelled reaction Zone in the vicinity of
`the underlying conductive element 16. Reactants such as
`enZyme, mediator and glucose move freely Within this Zone,
`but interfering species such as red blood cells containing
`oxygenated hemoglobin are excluded. This results in a
`device in Which the amount of current generated in response
`to a given amount of glucose varies by less than 10 percent
`over a hematocrit range of 40 to 60%, and Which is thus
`substantially insensitive to the hematocrit of the sample, and
`in fact performs substantially the same in blood as in a
`cell-free control solution. (FIGS. 3A—C, FIG. 4 and FIGS.
`5A—5C).
`Furthermore, the gelled reaction Zone presents a greater
`barrier to entry of blood analytes such as glucose Which
`makes the device diffusion, rather than kinetically limited.
`This leads to a device in Which the measured current varies
`by less than 10 percent over a temperature range from 20°
`C. to 37° C. and Which is thus essentially temperature
`independent. (FIGS. 6A and 6B)
`When making a glucose test strip, the integrated reagent/
`blood separation layer is advantageously formed from an
`aqueous composition containing 2 to 10% by Weight, pref
`erably 4 to 10% and more preferably about 4.5% of a binder
`such as hydroxyethylcellulose or mixtures of hydroxyeth
`ylcellulose With alginate or other thickeners; 3 to 10% by
`Weight, preferably 3 to 5% and more preferably about 4%
`silica; 8 to 20% by Weight, preferably 14 to 18% and more
`preferably about 16% of a mediator such as ferricyanide;
`and 0.4 to 2% by Weight, preferably 1 to 2% and more
`preferably about 1.6% of an enZyme such as glucose
`oxidase, assuming a speci?c activity of about 250 units/mg,
`or about 1000 to 5000 units per gram of ink formulation.
`The integrated reagent/blood separation layer may also
`include additional ingredients Without departing from the
`scope of the invention. For example, the nonconducting
`layer may include an antifoam. In addition, the nonconduct
`ing layer may be formulated With a buffering agent to control
`the pH of the reaction Zone. The pH may be maintained at
`a level Within the range from about pH 3 to pH 10. In one
`embodiment of the invention, it is of particular utility to
`maintain the pH of the device at a level above 8 because at
`this pH oxygen bound to hemoglobin is not released.
`Further, at this pH, the reaction rate of glucose oxidase With
`oxygen is very loW. Thus, selection of an appropriate pH can
`further stabiliZe the performance of the test strip against the
`effects of varying hematocrit. In an alternative embodiment
`of the invention, maintaining a loW pH (beloW pH 5.5, the
`optimium pH for reaction of glucose oxidase With oxygen)
`m