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`"EXPRESS MAIL"Mailing Label No. EL004276108US
`
`REISSUE PATENT
`
`First Named Inventor: Tomohiro Yamamoto,et al.
`Original Patent No.: 5,658,443
`Original Patent No. Issue Date: August 19,1997
`
`Attorney Docket
`No. 10059-042RE
`(P11856-01)
`
`PTOiall08
`U8,
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`
`Assistant Commissioner for Patents
`BOX PATENT APPLICATION
`Washington, D.C. 20231
`
`Transmitted herewith forfiling is the reissue utility patent application for U.S. Patent No.
`5,658,443 of Inventor(s):
`Tomohiro Yamamoto, Mariko Miyashita (nee Miyahara),
`Toshihiko Yoshioka, Satoko Tsuji (nee Fujisawa) and Shiro Nankai
`
`For:_BIOSENSOR AND METHOD FOR PRODUCING THE SAME
`
`Enclosed are:
`
`
`
`[X]
`
`Transmittal Letter.
`
`
`
`
`[X]
`
`[X]
`
`[X]
`
`Specification and claims (Amended) (16 Pages).
`
`_4_ sheets of drawings (photocopies of printed drawings) plus twocopies.
`Wehereby Request Transfer of Drawing from Original Patent Filed to this reissue
`application.
`
`[X]
`Reissue Oath/Declaration Under 37 C.F.R. § 1.175. (Unsigned)
`[XX]
`Offer to Surrender under 37 C.F.R. § 1.178 (signed).
`[X] Written Consent ofAssignee under 37 C.F.R. § 1.172 & Statement of Ownership
`Under 37 C.F.R. § 3.73(b).
`
`[X]
`
`Photocopy of U.S. Letters Patent 5,658,443, dated August 19, 1997.
`
`[]
`
`[]
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`[]
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`A certified copy of Application No.
`
`A verified statement to establish small entity status under 37 CFR § 1.9 and 37
`
`CFR § 1.27.
`
`Information Disclosure Statement.
`
`

`

`Thefiling fee has been calculated as shown below:
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`SMALL ENTITY P|
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`CLAIMS NO. FILED|NO. EXTRA BASIC FEE: BASIC FEE:
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`The Commissioneris hereby authorized to charge paymentofthe followingfees orcredit
`any overpayment to Deposit Account No. 50-1017. Twoadditional copiesofthis sheet are
`enclosed.
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`[X]___
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`The abovecalculated filing fee of $1,372.00
`Anyadditional fees required under 37 C.F.R. §1.16 or §1.17.
`Ifthe filing of any paper during the prosecution ofthis application requires an
`extension oftime in order for the paper to be timely filed, applicant hereby
`petitions for the appropriate extension oftime pursuant to 37 C.F.R. §1.136(a).
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`Respectfully submitted,
`
`WILLIAM W. SCHWARZE
`Registration No. 25,918
`AKIN, GUMP, STRAUSS, HAUER & FELD,L.L.P.
`One Commerce Square
`2005 Market Street - Suite 2200
`Philadelphia, PA 19103
`Telephone: (215) 965-1200
`Direct Dial: (215) 965-1270
`Facsimile: (215) 965-1210
`E-Mail: WSCHWARZE@AKINGUMP.COM
`
`(Date)
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`WWS:srn
`Enclosures
`
`PSJN2/248351.1
`
`

`

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`
`1
`BIOSENSOR AND METHOD FOR
`PRODUCING THE SAME
`
`BACKGROUND OF THE INVENTION
`
`1. Field of the Invention
`
`The present invention relates to a biosensor capable of
`rapidly quantifying a specific component in a sample solu-
`tion with high accuracy in a simplified manner, and to a
`method for producing the same.
`2. Description of the Related Art
`Varioustypes of biosensor have heretofore been proposed
`as a system for quantifying the specific component in the
`sample solution without requiring diluting or stirring of the
`sample solution.
`As an example of such biosensors, a glucose Sensor will
`be described in the following paragraphs. In general, a
`system combining glucose oxidase with an enzyme elec-
`trode or a hydrogen peroxideelectrode is already known as
`a method of quantifying glucose utilizing the enzyme elec-
`trode. The glucose oxidase selectively oxidizes a substrate,
`Le.,B-D-glucose into D-glucono-d-lactone by using oxygen
`as an electron acceptor. During this reaction, oxygen is
`reduced into hydrogen peroxide. By measuring the amount
`of the oxygen consumed in this reaction by an oxygen
`electrode, or by measuring the amount of the hydrogen
`peroxide produced in this reaction by a hydrogen peroxide
`electrode which utilizes a platinum electrode or the like, the
`glucose in the sample solution can be quantified.
`By the above-mentioned method,
`the measurement is
`however adversely influenced with a concentration of the
`dissolved oxygen depending on the subject of the measure-
`ment. Further, the measurement is made completely impos-
`sible under a condition lacking oxygen. A typeof the glucose
`sensor that does not use oxygen as the electron acceptor but
`uses a metal complex or an organic compound such as
`potassium ferricyanide, a derivative of ferrocene or a deriva-
`tive of quinone as the electron acceptor has therefore been
`developed. With this type of biosensor, by oxidizing a
`reductant of the electron acceptor produced as the result of
`the enzymereaction by the electrode, the concentration of
`the glucose can be determined based on the current con-
`sumed for this oxidation reaction. This manner of measure-
`mentis not limited to glucose but has been widely applied
`for the quantification of substrates other than glucose.
`Asan example of this type of biosensor, a glucose sensor
`is known (Japanese Laid-Open Patent Publication No. Hei
`1-114,747) which will be described below.
`The disclosed biosensor has a configuration comprising
`an electrical insulating base provided with an electrode
`system including a working electrode and a counter
`electrode, a filter layer composed of polycarbonate porous
`film, an electron acceptor carrying layer, an enzyme carrying
`layer, a buffer carrying layer, and a developing layer com-
`posed of woven cellulose, which are sequentially laminated
`on the insulating base by placing some space from the
`electrode system. In this configuration, the above-mentioned
`carrying layers are prepared by impregnating cellulosic
`porous films with aqueoussolutionsof the electron acceptor,
`the enzyme, and the buffer, and then drying the impregnated
`bodies.
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`The operation of this glucose sensor is as follows.
`The sample solution titrated on the developing layer is
`first passed to the buffer carrying layer. whereby the pH
`value of the sample solution is adjusted to a pH value that
`can give the highest activity to the enzymeby the buffering
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`action of the buffer. Next, the glucose in the sample solution
`reacts specifically with the glucose oxidase in the enzyme
`carrying layer. At the same time, the electron acceptor, such
`as potassium ferricyanide in the electron acceptor carrying
`layer, is reduced by the electron produced by the above-
`mentionedreaction to produce potassium ferrocyanide. The
`amountof the produced potassium ferrocyanide is directly
`proportional to the concentration of glucose contained in the
`sample solution. After the substances having a large molecu-
`lar weight such as protein which disturb the electrode
`reaction contained in the sample solution arefiltered off by
`the filter layer, the sample solution reaches the electrode
`system provided on the insulating base. In order to prevent
`erroneous measurement, part of the electrode system is
`covered with the insulating layer. By measuring the value of
`the current for oxidizing the potassium ferrocyanide pro-
`duced in the sample solution by the electrode system. it is
`possible to determine the glucose concentration of the
`sample solution.
`In the configuration of such prior art sensors, however.
`there is an inconvenience that an adverse influence is given
`to the responsive current, because wetting of the surface of
`the insulating base including the electrode system with the
`sample solution is not necessarily uniform and thus bubbles
`are retained between the porous bodyofthefilter layer and
`the insulating base. Further, if the sample solution contains
`substances liable to be absorbed in the electrode or sub-
`stances having an electrode activity, there would be a case
`wherein the response of the sensor is adversely influenced.
`As a method for overcoming the above-mentioned
`inconveniences, the following biosensor is proposed and
`disclosed in Japanese Laid-Open Patent Publication No. Hei
`2-062,952.
`In the disclosed configuration, the sensor comprises an
`electrically insulating base, an electrode system composed
`of a working electrode, a counter electrode and a reference
`electrode formed on the insulating base by meansof screen
`printing or the like, and a reaction layer including a hydro-
`philic polymer, an oxido-reductase, an electron acceptor, and
`a buffer as well if required, formed on the electrode system
`in a manner that the reactionlayeris in close contact with the
`electrode system.
`When the sample solution containing the substrate is
`titrated on the reaction layer, the reaction layer dissolves in
`the sample solution which is thereby adjusted to a pH value
`at which the highest enzyme activity is achieved by the
`buffering action of the buffer, the enzyme reacts with the
`substrate, and the electron acceptor is reduced. After the
`completion of the enzyme reaction, the reduced electron
`acceptor is electrochemically oxidized, and the concentra
`tion of the substrate contained in the sample solution is
`derived from the value Of the current consumed for oxidiz-
`ing the electron acceptor.
`Jn the above-mentioned configuration of the prior art
`sensor, if the biosensor is moistened, the buffer would be
`partly mixed with the enzyme to induce a chemical
`interaction, thereby lowering the enzymeactivity and dete-
`riorating the storing property of the biosensor.
`SUMMARY OF THE INVENTION
`It is therefore a primary object of the present invention to
`provide a biosensor that can be applied to quantification of
`a specific component contained in various biological
`samplesin a.rapid and simple manner with high accuracy.
`It is another object of the present invention to provide a
`biosensor that can be stored for a long period of time, and
`can be utilized in quality control of foodstuffs as well as in
`clinical tests.
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`Itis still another object of the present invention to provide
`a method for producing such biosensors while avoiding a
`possible mixing of an enzyme with a buffer during its
`manufacturing process.
`The present invention provides a biosensor comprising,
`an electrical insulating base,
`an electrode system including at least a working electrode
`and a counterelectrode which are provided on a face of
`the insulating base, and
`a reaction layer formed on the insulating base in close
`contact with the electrode system; wherein
`the reaction layer containsat least a hydrophilic polymer,
`an enzyme and a buffer, and
`the enzyme being separated from the buffer.
`In a preferred embodiment of the present invention, the
`reaction layer preferably comprises at least two layers,
`wherein a first layer is in contact with the electrode system
`and contains the enzyme and the hydrophilic polymer, and
`a secondlayer contains the buffer.It is also preferable for the
`second layer to comprise a lipid of amphipathic (lipophilic
`and hydrophilic) property.
`Tn another preferred embodiment of the present invention,
`the reaction layer preferably comprises at least two layers,
`wherein a first layer is in contact with the electrode system
`and contains the buffer and the hydrophilic polymer, and a
`second layer contains the enzyme.It is also preferable for
`the second layer to comprise a hydrophilic polymer being
`soluble in an organic solvent that does not dissolve the
`hydrophilic polymer contained in the first layer.
`It is preferable for the reaction layer of the biosensor in
`accordance with the present invention to contain an electron
`acceptor.
`Thepresent invention also provides a biosensor, wherein
`the reaction layer preferably comprises at least three layers,
`and a first layer contains the buffer and the hydrophilic
`polymer, and a second layer contains the enzyme and the
`hydrophilic polymer. It is also preferable for the second
`layer to further comprise an electron acceptor.
`Another preferred embodiment of the present invention
`further comprises a layer containing a lipid, especially an
`amphipathic lipid, placed to the outermost part of the
`reaction layer.
`In a further preferred embodiment of the present
`invention, the biosensor comprises a layer consisting essen-
`tially of a hydrophilic polymer placed in close contact with
`the electrode system.
`In still another preferred embodiment of the present
`invention, the layer containing the buffer and the hydrophilic
`polymer is in close contact with a layer containing the
`enzyme and the hydrophille polymer, wherein the hydro-
`philic polymers are different from each other, and wherein
`the hydrophilic polymer contained in the upper layer is
`soluble in an organic solvent that does not dissolve the
`hydrophilic polymer contained in the underlying layer.
`The present invention also provides a method for produc-
`ing a biosensor which comprises the steps of:
`forminga first layer containing an enzyme and a hydro-
`philic polymer by using water as the medium on a face
`of an insulating base in close contact with an electrode
`system including at least a working electrode and a
`counter electrode which are provided on the insulating
`base; and
`forming a second layer containing a buffer on the first
`layer by using an organic solvent as the medium that
`does not dissolve the hydrophille polymer.
`In a preferred embodiment of the above-mentioned
`method, the step of formingthe first layer comprises spread-
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`ing an aqueous solution which dissolves the enzyme and the
`hydrophilic polymer on the insulating base and drying the
`spread solution, wherein the step of forming the secondlayer
`comprises spreading a solution obtained by dispersing the
`buffer in an organic solvent solution of a lipid and drying the
`spread solution.
`In another preferred embodimentof the present invention,
`the step of forming the first layer comprises spreading an
`aqueoussolution which dissolves the enzyme and the hydro-
`philic polymer on the insulating base and drying the spread
`solution, wherein the step of forming the second layer
`comprises spreading a solution obtained by dispersing the
`buffer in an organic solvent solution of the hydrophilic
`polymer on the first layer and drying the spread solution.
`The present invention also provides a method for produc-
`ing a biosensor which comprises the stepsof:
`formingafirst layer containing a buffer and a hydrophilic
`polymer by using water as the medium on a face of an
`insulating base in close contact with an electrode
`system including at least a working electrode and a
`counter electrode provided on the insulating base; and
`forming a second layer containing a hydrophilic polymer
`and an enzyme on the first layer by using an organic
`solvent as the medium that does not dissolve the
`hydrophilic polymer contained in the first layer.
`In a preferred embodiment of the present invention, the
`step of forming the first
`layer comprises spreading an
`aqueous solution which dissolves the buffer and the hydro-
`philic polymeron the insulating base and drying the spread
`solution, wherein the step of forming the second layer
`comprises spreading an organic solvent solution of the
`hydrophilic polymer onthe first layer and drying the spread
`solution, and further dropping an aqueous solution of the
`enzymeon the second layer and drying the dropped solution.
`It is preferable that the above-mentioned aqueoussolution
`which dissolves the enzyme and the hydrophilic polymer
`further dissolves an electron acceptor.
`In the same manner,it is also preferable that the above-
`mentioned aqueous solution of the enzyme further dissolves
`an electron acceptor.
`Further, it is preferable that the method further comprises
`a step of forming a third layer by spreading an organic
`solvent solution of a lipid over the second layer and drying
`the spread solution.
`While novel features of the invention are set forth in the
`preceding,
`the invention, both as to organization and
`content, can be further understood and appreciated, along
`with other objects and features thereof, from the following
`detailed description and example when taken in conjunction
`with the attached drawings.
`BRIEF DESCRIPTION OF THE DRAWINGS
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`FIG. 1 is a cross-sectional side view showing an essential
`part of a biosensor prepared in accordance with Example 1
`of the present invention.
`FIG. 2 is an exploded perspective view of the biosensor
`shown in FIG. 1 removed of its reaction layer.
`FIG. 3 is a cross-sectional side view showing an essential
`part of a biosensor prepared in accordance with Example 2
`of the present invention.
`FIG.4 is a cross-sectional side view showing an essential
`part of a biosensor prepared in accordance with Example 3
`of the present invention.
`DETAILED DESCRIPTION OF THE
`PREFERRED EMBODIMENTS
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`In the following paragraphs, embodiments of the biosen-
`sor and method for producing the same in accordance with
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`the present invention will be described in detail with refer-
`ence to the attached drawings.
`As described in the above, the biosensor of the present
`invention has a configuration that the reaction layer formed
`on the electrode system on the insulating base is in close
`contact with the electrode system, and contains at least the
`hydrophilic polymer, the enzyme and the buffer, wherein the
`enzyme is separated from the buffer. Since the reaction layer
`contains the buffer, even in the case wherein the pH value of
`the sample solution does not coincide with the pH value
`whichgives the highest enzymeactivity, the pH value of the
`sample solution is automatically adjusted to the pH value
`which gives the highest activity to the enzyme when the
`sample solution reaches the buffer contained in the reaction
`layer. Therefore, there is no needfor previously adjusting the
`pH value of the sample solution with a buffer or the like and
`it is possible to measure the concentration of the specific
`component in the sample solution by a simple operation.
`Further, by separating the enzyme from the buffer in the
`reaction layer, itis possible to prevent a partial mixing of the
`buffer With the enzymeattributable to a possible wetting or
`moistening of the biosensor and a lowering of the activity of
`the enzymeattributable to the chemical interaction induced
`by the mixing, and thus to maintain the enzyme at a
`condition that stabilizes the enzyme during the storing
`period of the biosensor.
`In the biosensor prepared in accordance with the present
`invention, the layer containing the buffer is in close contact
`with the layer containing the enzyme, but the hydrophilic
`polymers contained in both layers are different from each
`other. By selecting the hydrophilic polymer contained in the
`upper layer as the one that is soluble in an organic solvent
`which does not dissolve the hydrophilic polymer contained
`in the underlying layer, a direct contact of the buffer with the
`enzymecan effectively be avoided during the manufacturing
`process of the biosensor.
`The biosensor having the above-mentioned. configuration
`can be obtained by the following manufacturing processes.
`Oneof the processes comprises the steps of formingafirst
`layer composed of the enzyme and the hydrophilic polymer
`on the insulating base, which is in close contact with the
`electrode system, by using water as a medium, and forming
`a second layer containing the buffer on the first layer by
`using an organic solvent as the medium that does not
`dissolve the hydrophilic polymer contained in thefirst layer.
`The other process comprises steps of forming a first layer
`composedof the buffer and the hydrophilic polymer on the
`insulating base, being in close contact with the electrode
`system, by using water as a medium, and forming a second
`layer composed of the enzyme and the hydrophilic polymer
`on the first layer by using an organic solvent as the medium
`that does not dissolve the first mentioned hydrophilic poly-
`mer.
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`It is preferable that the biosensor of the present invention
`has a layer containing a lipid that facilitates an infusion of
`the sample solution into the reaction layer. In addition to
`lecithin (phosphatidyl cholin) used in the following
`examples, an amphipathic (lipophilic and hydrophilic) lipid
`such as phospholipids, exemplified as phosphatidyl serine,
`phosphatidyl ethanolamine and thelike, are preferableas the
`lipid.
`As the hydrophilic polymer for forming the reaction layer,
`in addition to carboxymethyl cellulose and polyvinyl pyr-
`rolidone which are used in the following examples, there are
`exemplified polyvinyl alcohol, water soluble cellulose
`derivatives such as ethyl cellulose and hydroxypropyl cel-
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`lulose; gelatin, polyacrylic acid and its salts, starch andits
`derivatives, maleic anhydride and its salts, polyacrylamide,
`methacrylate resin, poly-2-hydroxyethyl methacrylate.
`Although the description on the following examples is
`limited to the two-electrode system composed only of the
`working electrode and the counter electrode, a more accurate
`measurement can be performed by employing a three-
`electrode system also including a reference electrode.
`In addition to potassium ferricyanide used in the follow-
`ing examples, p-benzoguinone, phenadine methosulfate and
`ferrocene can be used as the electron acceptor.
`As the buffer, any buffer that can demonstrate a pH value
`which gives the highest activity to the employed enzyme
`such as anysalts of citric acid can freely be used in addition
`to the phosphate buffer used in the examples.
`The present invention can widely be applied to any
`reaction system where an enzyme participates, such as
`alcohol sensor, sucrose sensor, and cholesterol sensor, in
`addition to the exemplified glucose sensor, lactic acid sensor
`and glucose sensor. In these cases, alcohol oxidase, lactic
`acid dehydrogenase, cholesterol oxidase, cholesterol
`dehydrogenase,xanthine oxidase, and an aminoacid oxidase
`can be used in compliance with the specific substance to be
`quantified, in addition to the fructose dehydrogenase,lactic
`acid oxidase and glucose oxidase.
`As described in the above, the biosensor of the present
`invention can be applied to the quantification of the specific
`component contained in the various biological samples in a
`rapid and simple manner with a high accuracy. Further, since
`the biosensor can be stored for a long period of time. its
`value ofutilization is great in quality control offoodstuffs as
`well as in clinical tests.
`
`EXAMPLE 1
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`(Fructose Sensor I)
`FIG. 1 is a cross-sectional side view showing a fructose
`sensor prepared in accordance with an embodiment of the
`present invention with its cover and a spacer omitted, and
`FIG. 2 is an exploded perspective view of the fructose sensor
`with its reaction layer omitted.
`An insulating base i is made of polyethylene terephtha-
`late. On the insulating base 1, there are provided lead wires
`2 and 3 ofsilver by means of screen printing. An electrode
`system including a working electrode 4 and a counter
`electrode 5 is also formed on the insulating base 1 by
`printing an electrically-conductive carbon paste containing a
`resin binder. Further, an insulating layer 6 is formed on the
`insulating base 1 by printing an insulating paste. The insu-
`lating layer 6 maintains areas of the exposed regionsof the
`working electrode 4 and the counter electrode 5 constant,
`and partly covers the lead wires 2 and 3.
`After the electrode region was prepared in this manner, a
`mixed aqueous solution composed of an aqueous solution
`(0.5 wt %) of a hydrophilic polymer, sodium salt of car-
`boxymethyl cellulose (hereinafter referred to CMC) which
`dissolved fructose dehydrogenase (EC1. 1. 99. 11.; herein-
`after referred to FDH) as an enzyme and potassium ferri-
`cyanide as an electron acceptor, was dropped on the elec-
`trode system. By being dried in a hotair dryer at 40° C. for
`10 minutes, an FDH-potassium ferricyanide-CMC layer 7
`was formed.
`On the FDH-potassium ferricyanide-CMC layer 7, there
`was dropped a dispersion prepared by dispersing microc-
`rystals of potassium dihydrogenphosphate and dipotassium
`hydrogenphosphateas a buffer in a toluene solution (0.5 wt
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`%) of lecithin as a dispersing medium, which wasthen dried
`to form a buffer-lecithin layer 8. Since toluene used as the
`solvent for forming the layer 8 did not dissolve CMC in the
`underlying layer, a direct contact of the buffer in the layer 8
`with the enzyme in the layer 7 was effectively avoided.
`Further, by the provision of the layer containing an amphi-
`pathic lipid such as lecithin on the surface of the reaction
`layer, an infusion of the sample solution from the surface
`into the reaction layer can be made with ease. As described
`in the above, the reaction layer of the fructose sensor was
`formed.
`The manufacturing process of the biosensor can be sim-
`plified by dropping the mixed solutions containing the
`hydrophilic polymer, the enzyme and the electron acceptor,
`each in a stroke, and by the subsequent drying. The tem-
`perature range during the drying step is preferably from 20°
`C. to 80° C. which does not lead to a deactivation of the
`enzyme butis sufficient for completing the drying in a short
`period of time.
`After forming the reaction layer in the above-mentioned
`manner, the fructose sensor was completed by adhering a
`cover 14 and a spacer 13 to the insulating base in a positional
`relationship shown by single dot-dash-lines in FIG. 2. By a
`simple operation of bringing the sample solution to a contact
`with a sample supplying inlet 15 provided on a tip of the
`sensor, the sample solution can easily be introduced into the
`reaction layer region. Since the supplying amount of the
`sample solution is dependent on the volume of a space
`formed by the cover 14 and the spacer 13, there is no need
`of measuring the supplying amount beforehand.
`Further, evaporation of the sample solution can be mini-
`mized during the measurement thereby enabling a measure-
`ment of high accuracy. In FIG. 2, a reference numeral 16
`designates an air inlet opening provided on the cover 14.
`Whena transparent resin is used as the material for consti-
`tuting the cover 14 and the spacer 13,it is possible to easily
`observe the condition of the reaction layer and the state of
`introducing the sample solution from the outside.
`Two minutes after supplying 3 pl of a fructose standard
`solution as the sample solution to the fructose sensor thus
`prepared through the sample supplying inlet 15, a pulse
`voltage of +0.5 V on the basis of the voltage at the counter
`electrode was applied to the working electrode. Then the
`anodic current value 5 seconds after the application was
`measured.
`When the sample solution reached the reaction layer, the
`sample solution dissolved the buffer-lecithin layer 8 to have
`a desirable pH value, and subsequently dissolved the FDH-
`potassium ferricyanide-CMC layer 7. During this process,
`the fructose contained in the sample solution was oxidized
`by the FDH, and then the potassium ferricyanide was
`reduced to a potassium ferrocyanide by shifting of electrons
`by the oxidation. Next, by the application of the above-
`mentioned pulse voltage, a current was generated for oxi-
`dizing the produced potassium ferrocyanide, and this current
`value corresponded to the concentration of fructose con-
`tained in the sample solution.
`The activity of the enzyme employed in the fructose
`sensor demonstrates its maximum value at pH 4.5 at 37° C.
`Since the fructose standard solution is substantially neutral,
`when the standard solution reaches the buffer-lecithin layer
`8, its pH value is adjusted to 4.5, thereby making the enzyme
`activity highest. Further, by separating the buffer from the
`enzyme,it is possible to improve the storing property of the
`sensor.
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`The response obtained with the thus prepared fructose
`sensorto the fructose standard solution demonstrates a linear
`
`
`

`

`8
`relationship for the fructose concentration, and the linear
`relationship can be maintained in storage for a long period
`of time.
`In the above-mentioned example. in place of the buffer-
`lecithin layer 8, another buffer-hydrophilic polymer layer
`may be formed by spreading a solution prepared by dispers-
`ing the buffer in a solution of a hydrophilic polymer dis-
`solved in an organic solvent which does not dissolve the
`CMCcontained in the underlying layer, such as an ethanol
`solution of polyvinyl pyrrolidone, followed by drying.
`EXAMPLE 2
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`
`
`
`
`(Fructose Sensor I)
`In a manner similar to that in Example 1, an electrode
`system composed of the working electrode 4 and the counter
`electrode 5 was formed on the insulating base 1 made of
`polyethylene terephthalate by means of screen printing, as
`shown by FIG. 3. By dropping an aqueous solution (0.5 wt
`%) of CMConthe electrode system and then drying, aCMC
`layer was formed. Next. an aqueous solution of the enzyme
`FDHandthe electron acceptor potassium ferricyanide was
`spread over the CMC layer and then dried to form an
`FDH-potassium ferricyanide-CMC layer 7. In this case
`however, the CMC, the FDH as weil as the potassium
`ferricyanide were partially mixed together and formed in a
`thin film of a thickness of several microns. That is, when the
`above-mentioned aqueous solution was dropped on the
`CMClayer, the previously formed CMC layer was once
`dissolved and then formed a layer 7 in a state partly mixed
`with the enzyme andthe like during the subsequent drying
`process.
`In this case however, since nostirring or the like operation
`was performed, a completely mixed state was not brought
`about but a state wherein the surface of the electrode system
`was covered only with the CMC wasbrought about by this
`process. Since the enzyme, the electron acceptorandthelike
`are prevented from a direct contact with the surface of the
`electrode system in this manner, it is considered that
`(i) there is a low possibility of an absorption of protein on
`the surface of the electrode system and a change in the
`characteristics of the electrode system by a chemical
`action of a substance having an oxidizing ability such
`as potassium ferricyanide, and
`Gi)as a result, it is possibile to obtain a sensor having a
`sensor response with high accuracy.
`On this FDH-potassium ferricyanide-CMC layer 7, a
`dispersion prepared by dispersing microcrystals of potas-
`sium dihydrogenphosphate and dipotassium
`hydrogenphosphate, as the buffer, in an ethanol solution of
`polyvinyl pyrrolidone (hereinafter referred to PVP) as the
`hydrophilic polymer in 0.5 wt % was dropped to cover the
`layer 7 completely, and then dried to forma buffer-PVP layer
`10. Since the ethanol employed in forming the layer 10 does
`not dissolve the CMC contained in the underlying layer. a
`direct contact of the enzyme in the layer 7 with the buffer
`contained in the layer 10 can effectively be avoided.
`By dropping a toluene solution of lecithin in 0.5 wt % on
`the buffer-PVP layer 10 and then drying the dropped
`solution, a lecithin layer 9 was formed on the layer 16. In the
`above-mentioned manner, a reaction layer of the fructose
`sensor shown in FIG. 3 was formed.
`By combining the insulating base formed with the reac-
`tion layer with a spacer 13 and a cover 14 shown by FIG. 2
`in a similar mannerto that in Example 1, the fructose sensor
`of this example was completed.
`Bythe provisionof the buffer-PVP layer 10, even in a case
`of selecting a fruit Juice and the like containing solid
`
`45
`
`50
`
`55
`
`65
`
`

`

`
`
`9
`components such as fruit flesh or pulp as the sample
`solution, a possible absorption of the above-mentioned flesh
`or pulp onthe surface ofthe electrode system and its adverse
`infiuence on the response of the sensor can effectively be
`prevented by this buffer-PVP layer, and at the sametime, the
`pH value of the sample solution can be made to a pH value
`that gives the maximum activity to the enzyme.
`The fructose sensor thus prepared demonstrates a rapid
`and a highly accurate response and has an excellent storing
`property because the buffer is separated from the enzyme as
`in Example 1.
`
`EXAMPLE 3
`
`(Lactic Acid Sensor)
`Tn a manner similar to that in Example 1, an electrode
`system was formed on the insulating base 1 made of
`polyethylene terephthalate by means of screen printing, as
`shown by FIG. 4. By dropping an aqueous solution (0.5 wt
`%) Of CMC, which aiso dissolved the buffer, potassium
`dihydrogenphosphate and dipotassium hydrogenphosphate,
`on the electrode system and then drying. a buffer-CMClayer
`11 was formed. Next, an ethanol solution (0.5 wt %) of PVP
`was spread over the buffer-CMClayer 11 sothat it covered
`the layer, and then dried to form a PVP layer. An aqueous
`solution of lactic acid oxidase (available from TOYOBO
`Co., Ltd., hereinafter referred to LOD) as an enzyme and
`potassium ferricyanide as an electron acceptor was spread
`over the PVP layer and then dried. In this case, however,
`since the PVP layer was partly dissolved in the above-
`mentioned aqueous solution. an LOD-potassium
`ferricyanide-PVP l