`
`EXHIBIT 1017
`
`US. PATENT NO. 5,171,689 TO KAWAGURI ET AL.
`
`lnfopia Ex. 1017 pg. 1
`
`

`

`
`
`IlllllllillllllllIllllllllllillllillllllllllllllllllllllllllllllllllllillll'
`U5005171689A
`.
`.
`5,171,689 -
`[11] Patent Number:
`[19]
`United States Patent
`
`Kawaguri et a1.
`‘
`[45] Date of Patent:
`Dec. 15, 1992
`
`[54] SOLID STATE BIOSENSOR
`[75]
`Inventors= 11:40:39 fixaggyiffuitaLShliqu
`an in;
`as i
`ljlma, ot o
`Hirakata, an of Japan
`
`4,45%242 5/1984 Zick et a1.
`........................... 128/23:
`4,45 , 8
`7/1984 C1 rk .....
`.. 128/
`
`4,467,811
`8/1984 C12rk ............................... 128/635
`
`.
`,
`OTHER PUBLICATIONS
`
`[73] Assignee: Matsushita Electric Industrial Co.,
`Ltd., Osaka, Japan
`
`Webster, Medical Instrumentation, Houghton Mifflin
`Co. (1978), see p. 540 FIG. 10.13.
`
`[21] Appl. No.: 339,698
`_
`[22] Filed:
`
`Apr. 18, 1989
`
`’
`
`[63]
`
`Related U.S. Application Data
`Continuation-impart of Ser. No. 932,945, Nov. 28,
`1986, abandoned, which is a continuation of Ser: No.
`673,751, Nov. 8, 1984, abandoned.
`
`.
`5
`Int. CL """""" CHM 1/38’ GOIN 27/26
`[51]
`[52] US. Cl. .................................... 435/290;,43S/288;
`435/817; 204/403; 128/635
`[58] Field of Search ................ 128/635; 435/817, 287,
`435/288, 290 204/403
`’
`
`,
`
`'
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`252/408 1
`3 791988 2/1974 Josef at al
`435/817
`435/817
`4,024,042
`5/1977 Enron 8‘ al‘ """
`..
`4,224,125 9/1980 Nakamura et a1.
`
`.. 128/635
`4,240,433 12/1980 Updike Ct 31.
`.. 435/817
`4,388,166 6/1983 Suzuki et a1.
`.....
`4,431,004 2/1984 .Bessman et a1.
`....................
`l28/635
`
`Primary Examiner—David L. Lacey
`Assistant Examiner—William K. Y. Chan
`Attorney, Agent, or Firm—Birch, Stewart, Ko1asch &
`Birch
`(31'
`ABS
`[57]
`.
`_
`.
`.
`As the conventional Simple bio-sensors for measuring
`the particular component in living bodies,
`there are
`those in which a change in dye caused by enzyme reac-
`tion is detected optically, but such bio-sensors had a
`h
`.
`.
`.
`.
`.
`f
`problem t at precismn is low owmg to disturbance o
`”‘5 C01“ °f l‘qmd “mp155'.W"h “9'55““ “5mg.”
`enzymeelectrode, the preCISion was high, but operation
`of measurement was troublesome. The present inven-
`tion made it possible to detect the concentration of the
`particular component electromechanically with rapid-
`ity, Simplicny and high preCiSion by merely putting 21
`porous substrate containing at least enzyme on the elec-
`trode system and impregnating the body with a liquid
`sample.
`
`17 Claims, 3 Drawing Sheets
`
`\
`
`lnfopia Ex. 1017 pg. 2
`
`

`

`US. Patent
`
`Dec. 15, 1992
`
`Sheét 1 of 3
`
`5,171,689
`
`
`
`
`Infopia Ex. 1017 pg. 3
`
`
`
`

`

`
`
`US. Patent
`
`Dec. 15, 1992
`
`Sheet 2 of 3
`
`5,171,689 .
`
`
`
`FIG. 5
`
`Response Currenl
`
`l uA)
`
`O
`
`200
`
`50 O
`
`Glucose Concentration (mg /d£)
`
`FIG. 6
`
`3O
`
`
`
`
`
`.
`D glucose
`(300mg/d1)
`
`Response Currenl (WM 20
`
`lO
`
`
`
`C glucose
`(I50 mg/di)
`
`
`
`
`
`20 4O 60 80 ICC 120 I40
`
`Amount Of Added Liquid Sample (111)
`
`Infopla Ex. 1017 pg. 4
`
`

`

`US. Patent
`
`Dec. 15, 1992
`
`Sheet 3 of 3
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`5,171,689
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`w 2
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`7
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`28
`
`FIG. 8
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`34
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`
` 32
`
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`' W
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`
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`'IIIIIIIIIIIJ"Ill/”I‘m
`/
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`33
`
`35
`
`
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`39
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`
`
`
`
`
`Ills-Illmlglllllt-Ilu
`gunman“;
`
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`V
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`38
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`43
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`36
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`,
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`Infopia Ex. 1017 pg. 5
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`
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`

`

`1
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`SOLID STATE BIO-SENSOR
`
`5,171,689
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`2
`method, the liquid sample can be used as it is without a
`necessity for pre-treatment, and therefore, the measure-
`ment became simple and precision was also improved.
`A case of glucose sensor will be taken as example. A
`flow-type sensor was developed as showu in FIG. 2 in
`which a liquid sample, e.g. blood or urine, is added to
`the sensor while applying a definite voltage to a glucose
`oxidase immobilized electrode 6 and passing a buffer
`solution 1 through a conduit 8 made of insulating mate-
`rials such as acrylic resin; glucose in the sample reacts ,
`with the immobilized glucose oxidase to produce hy-
`drogen peroxide which is then oxidized at the electrode
`6 to generate a current; and the glucose concentration
`of the sample can be detected'by measuring the strength
`of the current. The sensor of this type enables as many
`specimens as 200 to 300 per hour to be measured with
`rapidity and high precision, but it had a problem that
`the apparatus becomes large in size. The so-called
`batch-type sensor as shown in FIG. 3 was thus devel-
`oped in which aglucose oxidase immobilized electrode
`11 is placed in a vessel 10 which is then filled with a .
`buffer solution 12, and a liquid sample is added to the
`solution while stirring with a stirrer. The size of appara-
`tus could be made fairly small by using this type, but
`there occurred problems that a stirrer is essential, and
`that foaming and turbulent flow are caused by stirring
`to exert an adverse effect on the precision Also, there
`was a necessity to exchange the butTer solution and
`sometimes wash the electrode, and besides, because of
`the liquid sample being diluted with the buffer solution,
`precision was required for the amounts of buffer solu-
`tion and liquid sample.
`Thus, for simple measurement, a dry measurement-
`form sensor
`requiring no stirring apparatus is de-
`manded, and besides a high precision‘is required.
`DISCLOSURE OF THE INVENTION
`
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`CROSS-REFERENCE TO RELATED
`
`APPLICATION
`This application is a continuation-in-part of applica-
`tion Ser. No. 932,945 filed Nov. 28, 1986, now aban-
`doned, which is a continuation of application Ser. No.
`673,751, filed Nov. 8, 1984, now abandoned.
`TECHNICAL FIELD
`
`The present invention relates to a bio-sensor which
`makes it possible to determine the particular component
`of various biological samples with ease, rapidity and
`high precision.
`BACKGROUND OF THE INVENTION
`
`In recent years, various kinds of bio-sensor based on
`the specific catalytic action of enzyme have been devel-
`oped, and particularly, their application to clinical in-
`spection field is being tried.- At the present day wherein
`inspection items and the number of specimens are in-
`creasing, bio-sensors which enable rapid and high-preci-
`sion measurement are demanded.
`A case of glucose sensor will be taken as example. In
`these days of remarkable increase of diabetes, for mea-
`surement and control of blood sugar in blood, there is a
`demand for sensors applicable to tests on whole blood,
`because it takes a very long time to centrifuge blood and
`measure the separated plasma as conventionally carried
`out. There is provided a simple sensor like the test paper
`used for urinalysis, and this sensor comprises a stick-
`form support and a carrier put on the support contain-
`ing an enzyme which will react with glucose only and a
`dye which will change itself on enzyme reaction or by
`the product of the reaction. Blood is added to the car-
`rier, and a change in the dye after a definite period of
`time is measured visually or optically. But, the measure-
`ment is largely disturbed by dyes in blood, so that its
`precision is low.
`The multi-layer analytical carrier was thus proposed
`as shown in FIG. 1 (Japanese Utility Model Opening
`(KOKAI) No. 178495/ 1979). This carrier has a lami-
`nated structure wherein a reagent layer 2, developing
`layer 3, water-proof layer 4 and filter layer 5 are placed
`one upon another in this order on a transparent support
`1. When a blood sample is dropped down to the carrier,
`the blood is first freed from its solid components such as
`red blood cells, blood platelets, etc. on the filter layer 5,
`uniformly diffuses into the developing layer 3 through
`small pores in the‘water-proof layer 4 and comes into
`reaction in the reagent layer 2. After completion of the
`reaction, light is passed through the transparent support
`in the direction of an arrow to measure the substrate
`concentration spectroanalytically. Although this carrier
`is complicated in structure as compared with the con-
`ventional sample stick-form carrier, an improvement in
`precision was attained by removing blood cells, etc.
`But, it takes a long time for the penetration and reaction
`of blood, so that there was a necessity to put the water-
`proof layer 4 for preventing the sample from drying and
`to incubate at high temperatures for accelerating the
`reaction, which caused a problem of the apparatus and
`carrier becoming complicated,
`Thus, an electrode method comprising combining
`enzyme reaction with electrode reaction was developed
`as a simple method of good precision. Since there is no
`disturbance of colored substances in the electrode
`
`The bio-sensor of the present invention is composed
`of an insulating plate and an electrode system compris-
`ing a working electrode and a counter electrode put
`‘ thereon, said system being covered with a porous plate
`comprising at least one layer containing oxidoreduc-
`tase. This sensor is intended to measure the concentra-
`tion of substrates, e.g. urine sugar and blood sugar, by
`electrochemically detecting a reduced electron accep-
`tor produced when it is impregnated with a liquid sam-
`ple to carry out enzyme reaction, and also it is intended
`to measure the glucose concentration of fruitJuices.
`By using the bio-sensor of the present invention, meaw -
`surement can be carried out by merely adding a liquid
`sample directly to the porous plate containing at least
`enzyme without using a buffer solution. Also, since the -
`sample need not be diluted, there is no necessity to
`determine the sample and besides a trace amount of the
`particular component of the sample can be measured
`with simplicity and high sensitivity.
`
`45»
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`65
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`BRIEF EXPLANATION OF THE DRAWING
`FIG. 1 is a typical view illustrating one embodiment
`of the conventionally used glucose sensors.
`FIGS. 2 and 3 are typical views illustrating the con-
`ventional glucose sensor using an immobilized enzyme
`electrode.
`
`FIG. 41s a typical View illustrating one embodiment
`of the bio-sensor of the present invention.
`FIGS. 5 and 6 show the response characteristics of
`the bio-sensor shown in FIG. 4.
`
`lnfopia Ex. 1017 pg. 6
`
`
`
`

`

`3
`FIGS. 7, 8 and 9 are typical views illustrating other ,
`embodiments of the bio-sensor of the present invention.
`BEST FORM FOR PRACTICE OF THE
`INVENTION
`
`5,171,689
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`4
`rise at higher concentrations than this. The reason for
`this may be considered to be due to that supply of oxy-
`gen is not sufficient when the substrate concentration is
`high. But, measurement was possible at low concentra-
`tions of the substrate.
`
`Example 3
`
`Example 1
`Explanation will be given on a glucose sensor which
`is one of bio-sensors. Glucose oxidase was used as oxi-
`doreductase, and potassium ferricyanide was used as an
`oxidizing dye working in conjugation with the oxidore-
`ductase. FIG. 4 is a typical view illustrating one em-
`bodiment of a glucose sensor. Pieces of platinum wire of
`1 mm in diameter were buried in an insulating plate 19
`made of a polyvinyl chloride resin to prepare a working
`electrode 20, counter electrode 21 and reference elec-~
`trode 22, and nylon non-woven fabric 23 was put on the
`foregoing electrode system so as to cover the system.
`This nylon non-woven fabric 23 was prepared as fol-
`lows: 100 mg of glucose oxidase and 150 mg of pota-
`sium ferricyanide are dissolved in l c.c. of a phosphate
`buffer solution (pH 5.6), and nylon non-woven fabric is
`impregnated with the solution thus prepared and dried
`at room temperature.
`A standard, glucose solution, a liquid sample, was
`added to the nylon non-woven fabric 23 and after al-
`lowing the solution to well penetrate into the fabric,
`voltage was applied to the working electrode 20 with
`the reference electrode 22 as standard and varied at a
`rate of 0.1 V/sec so that it drew a saw-toothed line
`between 0 V and +0.5 V. For example, when the glu~
`cose in a sample is oxidized by glucose oxidase 24 car-
`ried on a nylon non-woven fabric 23, potassium ferricy-
`anide 25 is reduced by enzyme/dye conjugation reac-
`tion to produce potassium ferrocyanide. On oxidizing
`the potassium ferrocyanide by sweeping voltage ap-
`plied to the working electrode 20, anodic current flows.
`This anodic current is directly proportional
`to the
`amount of dye changed, and when the dye is present in
`sufficient amounts, since said amount corresponds to the
`substrate concentration, the concentration of glucose, a
`substrate, can be detected by measuring the strength of
`the anodic current. The relationship between the
`strength of peak current obtained and the concentration
`of glucose added showed a very good rectilinearity in
`the range of up to 500 mg/dl as shown by A in FIG. 5.
`The nylon non-woven fabric 23 was exchanged at every
`measurement, but reproducibility was good. Also, the
`amount of the standard glucose solution added was
`varied from 20 pl to 140 pl, but a definite value was
`obtained independently of the amount of the solution as
`shown by C and D in FIG. 6 when the glucose concen-
`tration was definite.
`
`Example 2
`
`The electrode system used in Example 1 was covered
`at the upper surface with nylon non-woven fabric car-
`rying glucose oxidase only, and then measurement was
`carried out in the same manner as in Example 1 with
`dropwise addition of the standard glucose solution. In
`this system, oxygen contained in air and the liquid sam-
`ple is used as the electron acceptor; when glucose reacts
`with glucose oxidase, hydrogen peroxide is produced in
`an amount corresponding to the glucose concentration
`and oxidized at the surface of the electrode; and the
`strength of anodic current generated by the oxidation is
`measured to obtain the glucose concentration. The peak
`of the current obtained, as shown by B in FIG. 5,
`showed a rectilinear rise up to 300 mg/dl but little or no
`
`FIG. 7 is a typical View illustrating another embodi-
`ment of the glucose sensor of the present invention. As
`shown in Example 1, measurement can stably be carried
`out if a three-electrode system comprising a working
`electrode, a counter electrode and a reference electrode
`is used, but it can also be carried out with a two-elec-
`trode system comprising a ‘working electrode and a
`counter electrode. Platinum was buried in an insulating
`plate 26 made of a polyvinyl chloride resin to prepare a
`. working electrode 27 and counter electrode 28. To
`stabilize the potential, the area of the counter electrode
`28 was made at least more than two times as large. as
`that of the working electrode 27. In the same manner as
`in Example 1, this electrode system was covered at the
`upper surface with nylon non-woven fabric 29 carrying
`glucose oxidase 30 and potassium ferricyanide 31, and
`measurement was carried out with dropwise addition of
`the standard glucose solution. It was found that the
`reproducibility was slightly poorer than in Example 1,
`but that glucose concentrations of up to 500 mg/dl
`could be measured. A silver/silver chloride (Ag/AgCl)
`counter electrode was used in place of the platinum one
`28, and it was found that the potential was stabilized,
`and that measurement could be carried out with good
`reproducibility if the area of this electrode was made
`equal to that of the electrode 27.
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`Example 4
`FIG. 8 is a sensor comprising an insulating plate 32
`made of a polyvinyl chloride resin and a working elec-
`trode 33, counter electrode 34 and reference electrode
`35 in a thin film form which were produced from plati-
`num on the plate by the spattering method. On these
`electrodes was placed the same nylon non~woven fabric
`carrying the enzyme and oxidizing dye as used in Exam-
`ple 1 so as to cover the region shown by a dotted line.
`Thereafter, response to the standard glucose solution
`was measured in the same manner as in Example 1 to
`obtain the same good response as in the electrode sys-
`tem in FIG.'4. Next, measurement was repeated using
`the standard glucose solution of the same concentration
`as well as the electrodes and nylon non-woven fabric
`newly exchanged at every measurement, and as a result,
`a very good reproducibility was obtained. Conse.
`quently, if the electrodes are produced by the spattering
`method or vacuum deposition method as in this exam~
`ple, electrodes having the same reSponse characteristics
`and any desired shape can be produced in large
`amounts, and by covering the electrodes with a porous
`plate carrying enzyme and oxidizing dye, high-perfor-
`mance bio-sensors which can be disposed after use, can
`be provided.
`
`Example 5
`
`65
`
`FIG. 9 is a typical view illustrating one embodiment
`of a glucose sensor. Platinum is buried in an insulating
`plate 36 made of a polyvinyl chloride resin to prepare a
`working electrode 37, counter electrode 38 and refer-
`ence electrode 43. Nylon non-woven fabric 39 is placed
`on the electrode system comprising these electrodes so
`
`lnfopia Ex. 1017 pg. 7
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`

`5,171,689
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`5
`as to cover the system. This nylon non—woven fabric 39
`is a one prepared through the same dissolution, impreg-
`nation and drying as in Example 1, arid it carries glucose
`oxidase 40 (oxidoreductase) and potassium ferricyanide
`41 (oxidizing dye), which works in conjugation with
`oxidoreductase, in a dry form. On the upper surface of
`the fabric 39 is placed a filter layer 42 comprising po-
`rous polycarbonate (pore diameter, 1 pm).
`On adding blood (whole blood) to this sensor, large.
`molecules in blood such as red blood cells were filtered
`off on the filter layer 42, and glucose in the blood came
`into reaction at the reaction layer comprising the nylon
`non-woven fabric 39 in the same manner as in Example
`1, whereby the glucose concentration could be detected
`at the electrode system. That is, from the strength of a
`current obtained, the glucose concentration could be
`detected based on a calibration curve previously pre-
`pared with a standard glucose solution. The current
`strength and glucose concentration showed a good
`correlation as compared with the conventionally used
`types of glucose sensor. The reaction layer comprising
`the enzyme and oxidizing dye and the filter layer were
`exchanged at every measurement, but the reproducibil-
`ity was good in either sample of standard glucose solu-
`tion and blood. Also, the amount of blood added was
`varied from 20 pl to 140 ill, but the amount of glucose
`showed a definite value independently of said amount
`since the dye and enzyme were present in sufficient
`amounts.
`
`By using the porous of polycarbonate as the filter
`layer 42, blood cells and viscous substances present in
`blood could previously be removed, and as a result,
`stains on the electrode could be decreased. Without the
`filter layer, blood cells adhered to the electrode during
`a long~terrn use to lower the strength of current ob-
`tained, so that there was necessity to wash the electrode
`with alcohol. But, by using the filter layer, it became
`possible to maintain the response with good reproduc-
`ibility by merely washing the electrode with water.
`Also,
`it was found that the porous plate which was
`useful to the above object was a one having a pore
`diameter within about 3 pm. Further when the porous
`substrate of polycarbonate was used after dipped in a
`1% aqueous solution of surface active agent, e.g. poly-
`ethylene glycol alkylphenyl ether (trade name: Triton
`X) and dried, filtration of blood became very fast with
`a further improved reproducibility. Hitherto, there was
`a problem that, since blood is fairly viscous, its filtration
`rate is low. But, by using a filter layer previously treated
`with surface active agents, filtration became fast and
`also reaction with enzyme and dye proceeded rapidly
`and uniformly to come to an end in as short a time as
`only about one minute after addition of a test sample.
`When the surface active agent was not used, it took
`about 1.5 minutes for the reaction to come to an end
`after addition of blood, and therefore the effect of the
`surface active agent to accelerate the measurement was
`large.
`As the surface active agent, polyoxyethylene glyc-
`erin fatty acid esters, polyoxyethylene alkyl ethers,
`polyethylene glycol fatty acid esters and the like can be
`used in addition to the foregoing example. By previous
`treatment of not only the filter layer but also the dye
`and enzyme with the surface active agent, the rates of
`filtration and penetration became fast to make it possible
`to accelerate the measurement.
`The reaction layer comprising the oxidizing dye and
`enzyme is preferably a hydrophilic porous membrane so
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`that it can absorb the liquid sample rapidly to advance
`the enzyme reaction. For example, when filter paper,
`non-Woven fabric of pulp, porous plate of ceramic or
`glass, etc. were used in addition to the nylon non~woven
`fabric, penetration of the liquid sample was quick: and
`uniform, and reproducibility was also good. Further,
`the rate of penetration of the sample into the above
`porous membrane was larger in the membrane pre-
`treated with the foregoing surface active agent than in
`the untreated membrane. The filter layer having an
`effect to accelerate the measurement may be placed on
`the electrode or the upper surface of the reaction layer.
`Penetration of the liquid sample was fastest when the
`filter layer was placed below the reaction layer, and the
`reaction time was also short. While, when the filter
`layer was placed on the reaction layer, there was an
`advantage that the reaction proceeds smoothly and a -
`high precision is obtained because the solid component
`of blood can first be filtered off so that there is no distur-
`bance of blood cells, etc. in the reaction layer. For the
`filter layer, use of non-woven fabrics, chemical fibers,
`paper (filter paper), etc. was thought of. But, the mate-
`rial component of blood cells could completely be ill-
`tered off by using membrane filter having a pore diame-
`ter of less than about 2—3 pm. Also, filtration could be
`carried out stepwise by laminating the membrane filter
`layer with the foregoing non-woven fabric, chemical
`fiber, paper or the like.
`In the above sensor, various methods could be em-
`ployed to carry glucose oxidase (enzyme) and potas-
`sium ferricyanide (dye). For example, it was also possi-
`ble to laminate two pieces of nylon non-woven fabric
`carrying the enzyme and dye, respectively. In carrying
`potassium ferricyanide on nylon non-woven fabric 39,
`potassium ferricyanide crystals on the fabric became
`much finer and more soluble in liquids by hot air-drying
`nylon non-woven fabric impregnated with a potassium
`ferricyanide solution than by drying the fabric at room
`temperature. The potassium ferricyanide crystal on the
`fabric became further finer when the nylon non-woven
`fabric impregnated with a potassium ferricyanide solu-
`tion was dipped in an organic solvent having a great
`solubility in water, for example ethanol, and vacuum— .
`dried. By carrying potassium ferricyanide in a finely
`pulverized form, even a highly viscous sample such as
`blood completed the reaction within one minute with an
`improved reproducibility. Since the enzyme is poorly
`resistant to heat, etc., it was dried at room temperature
`after dipped in the above organic solvent. Also, when
`both the enzyme and oxidizing dye are carried on one
`piece of porous plate, the dye and enzyme could be
`carried in a very soluble form by dipping the porous
`plate carrying a color and an enzyme ethanol and vacu~
`um-drying. Further, the enzyme may be immobilized by
`crosslinldng with glutaraldehyde after dipping, and it
`could be stored stably for a long time by such immobi1i~
`zation.
`
`Example 6
`
`In Example 5, a porous membrane such as nylon
`non-woven fabric was used as a porous substrate, but in
`addition to this, inorganic carriers such as SiOz also may
`be used in the form of a porous plate produced by press-
`molding. When $02 was pressure-molded into a size of
`7 mm (diameter))<l mm (thickness) by 5 t/cm2 pres-
`sure, impregnated with an aqueous solution containing
`glucose oxidase and potassium ferricyanide and then
`dried, penetration of blood into this molded SiOz was a
`
`Infopia Ex. 1017 pg. 8 .
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`determined by using an alcohol oxidase (alcohol sensor)
`instead of a glucose oxidase.
`What is claimed is:
`1. A bio-sensor for measuring a substrate concentra-
`tion of a liquid sample, comprising:
`at least a working electrode and a counter electrode,
`an insulating substrate plate which supports and insu-
`lates the working electrode and the counter elec-
`trode from one another, and
`a porous plate fixed on the insulating substrate plate,
`wherein said porous plate carries at least an oxido-
`reductase and a buffer, the oxidoreductase and the
`buffer are in a dry state, and wherein the bio-sensor
`is constructed so as not to contain a liquid contain-
`ing chamber.
`2. The bio-sensor according to claim 1, wherein the
`buffer is a phosphate.
`3. The bio-sensor according to claim 1, wherein an
`electron acceptor is carried on the porous substrate in a
`dry state.
`4. The bio-sensor according to claim 1 further com-
`prises an oxidizing dye.
`5. The bio-sensor according to claim 4, wherein the
`oxidizing dye is a member selected from the group
`consisting of potassium ferricyanide, p-benzoquinone,
`2,6-dichlorophenolindophenol, methylene. blue, phen-
`azine methosulfate and potassium beta-naphtoquinone-
`4-sulfonate.
`6. The bio-sensor according to claim 1, wherein the
`oxidoreductase is a member selected from the group
`consisting of glucose oxidase, alcohol oxidase, xanthine
`30 oxidase and cholesterol oxidase.
`7. The bio-sensor according to claim 1, wherein the
`oxidoreductase is glucose oxidase.
`8. The bio-sensor according to claim 1, wherein the
`working electrode comprises platinum.
`9. The bio—sensor according to claim 8, wherein the
`porous plate has a pore diameter of less than 3 micron
`meters.
`»
`
`7
`little slower than in the nylon non-woven fabric. Since,
`however, the porous membrane of polycarbonate also
`can be produced at the same time by pressure~molding,
`the filter layer and reaction layer could integrally be
`molded to simplify their production. Further,
`it was
`also possible to finely pulverize glucose oxidase and
`potassium ferricyanide and after mixing and adding a
`small amount of $02, to pressure~mold the mixture. In
`this case, since the glucose oxidase and potassium ferri-
`cyanide rapidly dissolve in blood to form a uniform
`mixture, the reaction became very fast.
`
`Example 7
`In Example 5, blood was filtered using a filter layer
`treated with a surface active agent. In this example,
`however, this treated filter layer was further impreg-
`nated with 10 mg/c.c. of a sodium fluoride (anticoagu-
`lant) solution and dried, and the procedure of Example
`5 was repeated but using the filter layer thus obtained.
`As a result, blood could be filtered in only ten seconds
`without coagulation, and besides measurement was
`completed in as short a time as only 50 seconds after
`addition of blood. Thus, this method made a great con-
`tribution to acceleration of measurement.
`.
`As the anticoagulant, sodium fluoride is suitable be-
`cause it is stable and simple in handling. But, heparin,
`sodium citrate, ethylenediamine tetraacetic acid, etc.
`were also useful to carry out blood filtration rapidly.
`As the dye, potassium ferricyanide used in the forego-
`ing examples is suitable because it reacts stably, but
`p-benzoquinone is suitable for acceleration because it is
`fast in reaction rate. Further, 2,6-dichlorophenolindo-
`phenol, methylene blue, phenazine methosulfate, potas.
`sium B-naphthoquinone-4-sulfonate, etc. may also be
`used.
`
`The sensors in the foregoing examples can be applied
`to not only glucose but also systems in which oxidore-
`ductase takes part such as alcohol sensors, cholesterol
`sensors and the like. Glucose oxidase was used as the
`oxidoreductase, but other enzymes such as alcohol oxi-
`dase, xanthine oxidase, cholesterol oxidase, etc. may
`also be used.
`
`Possibility of Application to Industry
`The bio~sensor of the present invention makes it pos-
`sible to measure the particular component in various
`biological samples with rapidity, high precision and
`simplicity, so that its utility value in clinical inspection is
`very large.
`The porous plate fixed on the insulating substrate
`plate has a pore size which is effective for retaining a
`liquid sample of body fluids such as blood, urine, saliva,
`lympha, sweat, tears and the like and other liquid sam-
`ples such as wine, fruit juices or glucose solution. More-
`over, any biological sample containing a specific sub-
`strate to be measured may be fixed on the porous plate
`so long as the porous plate has a pore size effective for
`retaining the sample. Preferably, the pore size of the
`plate is between 1000 Angstroms and 1 mm.
`The bio-sensor of the present invention is applicable
`to various fields such as biologicals, foods, industrials,
`etc. Further, the bio-sensor can be used for determining
`alcohol concentration, cholesterol concentration, etc.
`other than glttcose concentration in a liquid phase. One
`of ordinary skill in the art can readily understand that
`the glucose content or cholesterol content in lympha,
`for instance, can be determined according the inven-
`tion. Examples of food or’industrial fields include fruit
`juices or glucose solution, but similar liquid biological
`materials can be determined in a similar manner. For
`instance,
`the alcohol concentration in wine may be
`
`
`
`
`35
`
`10. The bio-sensor according to claim I, further com-
`prises a reference electrode.
`11. The bio-sensor according to claim 1, wherein the
`porous plate is a hydrophilic porous membrane.
`12. The bio-sensor according to claim 1, further com-
`prising a filter having a pore size of less than 3 micron
`meters positioned and arranged on the porous plate.
`13. The bio-sensor according to claim 12, wherein an
`45 anticoagulant is carried on the filter.
`14. The bio-sensor according to claim 12, which fur-
`ther comprises a reference electrode.
`.
`15. The bio-sensor according to claim 1, wherein the
`g counter electrode comprises platinum or silver/silver
`50 chloride.
`16. The bio-sensor according to claim 1, wherein the
`pore size of the porous plate is between 1000 Angstroms
`and 1 mm.
`17. A bio-sensor for measuring a substrate concentra-
`55 tion of a liquidrsample, which comprises:
`at least a working electrode and a counter electrode,
`an insulating substrate plate which supports and insu-
`lates the working electrode and the counter elec-
`trode from one another, and
`a porous plate fixed on the insulating substrate plate,
`wherein said porous plate carries at least an oxido-
`reductase and a buffer, the oxidoreductase and the
`buffer are in a dry state and contacts the electrodes
`without an intervening electrolyte layer between
`(i) the oxidoreductase and the buffer and (ii) the
`electrodes, and wherein the bio-sensor is con-
`structed so as not to contain a liquid containing
`chamber.
`#
`t
`t
`i
`#
`
`60
`
`65
`
`Infopia Ex. 1017 pg. 9
`
`