United States Patent [19]
`Ikeda et al.
`
`llllllllllllllllllllllIlllllllllllllllllllllllllllllIllllllllllllllllllllll
`5,582,697
`Dec. 10, 1996
`
`USOO5582697A
`[11] Patent Number:
`[45] Date of Patent:
`
`[54] BIOSENSOR, AND A NIETHOD AND A
`DEVICE FOR QUANTIFYING A SUBSTRATE
`IN A SAMPLE LIQUID USING THE SAlVIE
`
`[75] Inventors: Shin Ikeda, Katano; Toshihiko
`Yoshioka, Osaka; Shiro Nankai,
`Hirakata; Haruhiro Tsutsumi,
`Ehime-ken; Hideyuki Baba,
`Matsuyama; Yoshinobu Tokuno,
`Matsuyama; Syoji Miyazaki,
`Matsuyama, all of Japan
`
`[73] Assignee: Matsushita Electric Industrial Co.,
`Ltd., Osaka, Japan
`
`[21] Appl. No.: 425,820
`[22] Filed:
`Apr. 20, 1995
`[30]
`Foreign Application Priority Data
`
`Mar, 17, 1995
`
`[JP]
`
`Japan .................................. .. 7-058939
`
`[51] Int. Cl.6 ................................................... .. G01N 27/26
`[52] US. Cl. ........................ .. 204/403; 204/415; 204/412;
`204/406; 435/817; 435/287.9; 205/778;
`205/777.5
`
`[58] Field of Search ................................... .. 204/403, 415,
`204/412, 153.12, 406; 435/817, 288, 291
`References Cited
`U.S. PATENT DOCUMENTS
`
`[56]
`
`4,172,770 10/1979 Semersky et al. .................... .. 204/412
`5,192,415
`3/1993 Yoshioka et al.
`.. 204/403
`5,264,103 11/1993 Yoshioka et al. ..................... .. 204/403
`Primary Examiner—Bruce F. Bell
`Attorney, Agent, or Finn-Renner, Otto, Boisselle & Sklar
`[57]
`ABSTRACT
`
`The biosensor of this invention can quantify a substrate in a
`sample liquid by electrochemically measuring the amount of
`an electron acceptor that has been reduced by electrons
`generated in a reaction between the substrate and an oxi
`doreductase. The biosensor has an electrically insulating
`substrate and an electrode system formed on the substrate
`including a working electrode, a counter electrode and a
`third electrode used for detecting a liquid junction. The third
`electrode can be used merely for detecting a liquid junction,
`or can be used as both a reference electrode and a liquid
`junction detecting electrode.
`
`16 Claims, 4 Drawing Sheets
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`LIFESCAN SCOTLAND LTD. EXHIBIT 2006
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`US. Patent
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`Dec. 10, 1996
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`Sheet 1 of 4
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`5,582,697
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`FIG. I
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`U.S. Patent
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`Dec. 10, 1996
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`Sheet 2 of 4
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`5,582,697
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`

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`US. Patent
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`Dec. 10, 1996
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`Sheet 3 of 4
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`5,582,697
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`FIG. 5
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`FIG. 6
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`0.5V
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`FIG. 7
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`US. Patent
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`Dec. 10, 1996
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`Sheet 4 0f 4
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`5,582,697
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`5,582,697
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`1
`BIOSENSOR, AND A METHOD AND A
`DEVICE FOR QUANTIFYING A SUBSTRATE
`IN A SAMPLE LIQUID USING THE SAME
`
`BACKGROUND OF THE INVENTION
`
`1. Field of the Invention:
`The present invention relates to a biosensor that can easily
`quantify a substrate (a speci?c component) in a sample
`liquid with speed and accuracy, and to a method and a device
`for quantifying a substrate by using the biosensor. More
`particularly, the invention relates to a biosensor that can
`quantify a substrate in a sample liquid by electrochemically
`measuring the amount of an electron acceptor that has been
`reduced with an electron generated in a reaction between the
`substrate in the sample liquid and an oxidoreductase that
`speci?cally reacts with the substrate, and to a method and a
`device for quantifying a substrate by using the biosensor.
`2. Description of the Related Art:
`The optical rotation method, the colorimetric method, the
`reductimetry method and other methods using different
`kinds of chromatographies have been developed as methods
`for quantitative analysis of saccharides such as sucrose and
`glucose. However, none of these methods can provide high
`accuracy due to the relatively low speci?city against sac
`charides. Among these methods, the optical rotation method
`is easy to operate but is largely in?uenced by the operating
`temperature. Therefore, it is not appropriate for common use
`at home and the like.
`Various types of biosensors utilizing a speci?c catalysis of
`an enzyme have been recently developed. As an example of
`methods for quantifying a substrate in a sample liquid, a
`method for quantifying glucose will now be described. For
`electrochemically quantifying glucose, a method using glu
`cose oxidase (ECl.l.3.4; hereinafter referred to as GOD)
`and an oxygen electrode or a hydrogen peroxide electrode is
`generally known (for example, “Biosensor” edited by Shui—
`chi Suzuki, Kodansha Kabushiki Kaisha).
`GOD selectively oxidizes B-D-glucose into D-glucono
`S-lactone by using oxygen as an electron acceptor. In an
`oxidation reaction using GOD in the presence of oxygen,
`oxygen is reduced to hydrogen peroxide. In the aforemen
`tioned method, the amount of reduced oxygen is measured
`by using an oxygen electrode, or the amount of increased
`hydrogen peroxide is measured by using a hydrogen perox
`ide electrode. Since the amounts of the reduced oxygen and
`the increased hydrogen peroxide are in proportion to the
`content of glucose in the sample liquid, glucose can be
`quanti?ed based on the amount of the reduced oxygen or the
`increased hydrogen peroxide.
`The above-mentioned method, however, has a problem in
`that the measurement result is largely affected by the con
`centration of oxygen in the sample liquid, as can be pre
`sumed from the reaction process. Furthermore, when no
`oxygen is contained in the sample liquid, the above-men
`tioned method cannot be adopted.
`In order to solve this problem, a new type of glucose
`sensor has been developed, in which an organic compound
`such as potassium ferricyanide, a ferrocene derivative and a
`quinone derivative or a metal complex is used as an electron
`acceptor instead of oxygen. In this type of sensor, the
`reduced form of an electron acceptor resulting from the
`enzymatic reaction is oxidized on an electrode, and the
`concentration of glucose contained in a sample liquid is
`obtained based on the amount of the measured oxidation
`current. By using an organic compound or a metal complex
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`as an electron acceptor instead of oxygen, it is possible to
`hold a known amount of GOD and its electron acceptor on
`an electrode stably and accurately, so as to form a reaction
`layer. In this case, the reaction layer can be integrated with
`the electrode in a substantially dry state. Therefore, a dis
`posable glucose sensor based on this technique has recently
`come to public notice. In this disposable glucose sensor, the
`concentration of glucose in a sample liquid can be easily
`measured with a measuring device merely by introducing
`the sample liquid into the sensor removably connected to the
`measuring device. Such a method can be applied not only to
`the quanti?cation of glucose but also to the quanti?cation of
`any other substrate contained in a sample liquid.
`When a substrate in a sample liquid is quanti?ed by such
`a method, a two-electrode system sensor having a working
`electrode and a counter electrode is generally used.
`In the measurement using such a two-electrode system
`sensor, before applying a voltage between the working
`electrode and the counter electrode to obtain a current
`response, a voltage is generally applied between the working
`electrode and the counter electrode so as to detect a liquid
`junction based on the change of a resistance value between
`these electrodes. In such a case, the interface between the
`electrodes can be varied by the application of the voltage,
`resulting in affecting the measurement. Furthermore, the
`resistance value between the working electrode and the
`counter electrode can be sometimes varied to start the
`measurement before supplying a su?icient amount of a
`sample liquid to the electrode system. This also affects the
`measurement.
`
`SUMMARY OF THE INVENTION
`
`The biosensor of this invention quanti?es a substrate in a
`sample liquid by electrochemically measuring an amount of
`an electron acceptor that has been reduced by electrons
`generated in a reaction between the substrate and an oxi
`doreductase. The biosensor comprises an electrically insu
`lating substrate, an electrode system formed on the substrate
`including a working electrode, a counter electrode and a
`third electrode used for detecting a liquid junction, and a
`reaction layer that is formed over at least the working
`electrode and the counter electrode of the electrode system
`and includes the oxidoreductase.
`In one embodiment, the third electrode is disposed farther
`from a sample supply port than the working electrode and
`the counter electrode, so that a sample liquid supplied
`through the sample supply port reaches the third electrode
`after reaching the working electrode and the counter elec
`trode.
`In another embodiment, the third electrode is disposed
`nearer to a sample supply port than the counter electrode, so
`that a sample liquid supplied through the sample supply port
`reaches the third electrode before reaching the working
`electrode.
`In another embodiment, the counter electrode includes a
`main electrode portion formed in substantially a C-shape in
`a plane view and an opening formed in the main electrode
`portion, the working electrode disposed inside of the counter
`electrode so as to be electrically insulated from each other,
`and a lead connected to the working electrode is led from the
`inside to the outside of the counter electrode through the
`opening.
`In another embodiment, a peripheral portion of the
`counter electrode is opened to provide an electrode receiving
`
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`3
`portion, and the third electrode is disposed within the
`electrode receiving portion.
`In another embodiment, the counter electrode includes a
`main electrode portion formed in substantially a C-shape in
`a plane view and an opening formed in the main electrode
`portion, the working electrode disposed inside of the counter
`electrode so as to be electrically insulated from each other,
`a lead connected to the working electrode is led from the
`inside to the outside of the counter electrode through the
`opening, and the third electrode is disposed between the
`counter electrode and the working electrode so as to be
`electrically insulated from one another.
`In another embodiment, the reaction layer is formed also
`over the third electrode.
`In another embodiment, a cover is provided over the
`substrate, and a space serving as a sample supply path is
`formed between the substrate and the cover.
`In another embodiment, the electrode system is exposed
`to the space.
`In another embodiment, the reaction layer further includes
`an electron acceptor.
`’
`In another embodiment, the reaction layer further includes
`a hydrophilic polymer.
`The method for quantifying a substrate in a sample liquid
`of this invention uses the aforementioned biosensor. This
`method comprises the steps of applying a voltage between
`the counter electrode and the third electrode, supplying a
`sample liquid to the reaction layer, detecting an electrical
`change between the counter electrode and the third electrode
`caused by supplying the sample liquid to the reaction layer,
`applying a voltage between the working electrode and both
`of the third electrode and the counter electrode or the counter
`electrode after detecting the electrical change, and measur
`ing a current ?owing between the counter electrode and the
`working electrode after applying the voltage.
`The quantifying device for a substrate contained in a
`sample liquid of this invention comprises the aforemen
`tioned biosensor, means for detecting an electrical change
`between the counter electrode and the third electrode caused
`by supplying a sample liquid to the reaction layer, means for
`applying a voltage between the working electrode and both
`of the third electrode and the counter electrode or the counter
`electrode after detecting the electrical change, and means for
`measuring a current ?owing between the working electrode
`and the counter electrode.
`In another embodiment, the quantifying device for a
`substrate contained in a sample liquid, includes the afore
`mentioned biosensor and a measuring device removably
`connected to the biosensor. In this device, the measuring
`device includes: current/voltage converting circuits con
`nected to the third electrode of the biosensor; A/D convert
`ing circuits connected to the current/voltage converting
`circuits; current/voltage converting circuits capable of being
`connected to the working electrode of the biosensor via a
`switch; AID converting circuits connected to the current/
`voltage converting circuits; and a controller connected to the
`respective A/D converting circuits, the switch being turned
`ON or OFF with respect to the working electrode by a
`control of the controller. Also in this device, an electrical
`change between the counter electrode and the third elec
`trode, caused by a supply of a sample liquid to the reactive
`layer, is detected by the control portion under a condition
`that the switch is insulated from the working electrode, and
`then a voltage is applied between the working electrode and
`both of the third electrode and the counter electrode or the
`counter electrode under a condition that the switch is con
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`nected to the working electrode and a current ?owing
`between the working electrode and the counter electrode is
`measured.
`In another embodiment, the quantifying device for a
`substrate contained in a sample liquid, includes the afore
`mentioned biosensor and a measuring device removably
`connected to the biosensor. In this device, the measuring
`device includes: current/voltage converting circuits switch
`ably connected to the third electrode of the biosensor and the
`working electrode; A/D converting circuits connected to the
`current/voltage converting circuits; a controller connected to
`the respective A/D converting circuits, the switch being
`alternatively connected to the working electrode and the
`third electrode by a control of the controller. Also in this
`device, an electrical change between the counter electrode
`and the third electrode, caused by a supply of a sample liquid
`to the reactive layer, is detected by the control portion under
`a condition that the switch is connected to the third elec
`trode, and then a voltage is applied between the working
`electrode and the counter electrode under a condition that
`the switch is connected to the working electrode and a
`current ?owing between the working electrode and the
`counter electrode is measured.
`Thus, the invention described herein makes possible the
`advantages of (1) providing a biosensor that can easily
`quantify a speci?c substrate in a sample liquid with speed
`and accuracy; (2) providing a biosensor in which the inter
`face between a counter electrode and a working electrode is
`not varied in detecting a liquid junction, so that the detection
`of the liquid junction does not affect the measurement; (3)
`providing a biosensor in which a potential at a counter
`electrode used as a reference is not varied by an oxidation/
`reduction reaction at a working electrode, so as to decrease
`errors and deviation in the measurement; and (4) providing
`a biosensor that can quantify saccharides in fruit or saccha
`rides in blood, lymph, urine and saliva or other body ?uids.
`These and other advantages of the present invention will
`become apparent to those skilled in the art upon reading and
`understanding the following detailed description with refer
`ence to the accompanying ?gures.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIG. 1 is an exploded perspective view of a glucose
`sensor as an example of a biosensor of this invention from
`which a reaction layer is removed;
`FIG. 2 is a plane view of a substrate having an electrode
`system used in a glucose sensor as another example of the
`biosensor of this invention;
`FIG. 3 is a circuit diagram for the biosensor of FIG. 2 and
`a measuring device connected thereto;
`FIG. 4 is a plane view of a substrate having an electrode
`system used in a glucose sensor as still another example of
`the biosensor of this invention;
`FIG. 5 is a plane view of a substrate having an electrode
`system used in a glucose sensor as still another example of
`the biosensor of this invention;
`FIG. 6 is a plane view of a substrate having an electrode
`system used in a glucose sensor as still another example of
`the biosensor of this invention;
`FIG. 7 is an exempli?ed circuit diagram for the biosensor
`of this invention and a measuring device connected thereto;
`and
`FIG. 8 is another exempli?ed circuit diagram for the
`biosensor of this invention and a measuring device con
`nected thereto.
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`5,582,697
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`5
`DESCRIPTION OF THE PREFERRED
`EMBODIMENTS
`
`A biosensor of this invention comprises an electrically
`insulating substrate, an electrode system that is formed on
`the substrate and includes a working electrode, a counter
`electrode and a third electrode, and a reaction layer formed
`over the working electrode and the counter electrode of the
`electrode system. The working electrode, the counter elec
`trode and the third electrode of the electrode system are
`preferably formed on the same plane on the substrate.
`The electrically insulating substrate can be formed from a
`plate made of a synthetic resin such as polyethylene tereph
`thalate or any other suitable resin known in the art.
`The electrode system including the working electrode, the
`counter electrode corresponding to the working electrode
`and the third electrode can be formed on the substrate by a
`known method. For example, after forming leads on the
`substrate, the working electrode, the counter electrode and
`the third electrode are formed so as to be connected to the
`respective leads and be insulated from one another. The
`material for the leads and the electrodes can be of any known
`conductive materials such as silver paste and carbon paste.
`Furthermore, a silver/silver chloride electrode can be used.
`The shapes and the positions of the respective electrodes
`of the electrode system can be variously modi?ed. For
`example, the counter electrode can be in the shape of a ring
`or be substantially a C-shape in a plane view. The working
`electrode can be positioned in a space within the ring-shaped
`or C-shaped counter electrode so as to be electrically insu
`lated therefrom. By providing the working electrode inside
`of the counter electrode, these electrodes can be positioned
`adjacent to each other. In this case, the reaction layer
`covering the working electrode and the counter electrode
`can be easily formed.
`When the counter electrode is formed so as to have a
`substantially C-shaped main electrode portion and an open
`ing formed in the main electrode portion, a lead connected
`to the working electrode positioned inside of the main
`electrode portion is led from the inside to the outside of the
`main electrode portion through the opening.
`In a peripheral portion of the counter electrode can be
`formed an electrode receiving portion for the third electrode.
`The shapes of the electrode receiving portion and the third
`electrode can be variously modi?ed according to the
`required application. Furthermore, the position of the elec—
`trode receiving portion in the counter electrode can be varied
`depending upon the application of the biosensor.
`For example, the third electrode can be positioned farther
`from a sample supply port of the biosensor than the working
`electrode and the counter electrode, so that a sample liquid
`supplied through the sample supply port can reach the third
`electrode after reaching the working electrode and the
`counter electrode. Alternatively, the third electrode can be
`positioned nearer to the sample supply port than the working
`electrode, so that a sample liquid supplied through the
`sample supply port can reach the third electrode before
`reaching the working electrode and the counter electrode.
`When the third electrode is positioned farther from the
`sample supply port than the .working electrode and the
`counter electrode, the change of a resistance value between
`the counter electrode and the third electrode cannot be
`detected until a space including all of the three electrodes is
`?lled with a sample liquid. Accordingly, by utilizing such a
`third electrode for detecting a liquid junction, it can be
`de?nitely determined whether or not a sample liquid sup
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`plied through the sample supply port has covered the entire
`reaction layer.
`To the contrary, when the third electrode is positioned
`nearer to the sample supply port than the working electrode
`and the counter electrode, the third electrode is soaked with
`the supplied sample liquid without fail. Accordingly, by
`utilizing such a third electrode as a reference electrode, a
`reference potential can be more stabilized, resulting in
`attaining a measurement having less deviation.
`The third electrode can also be positioned between the
`counter electrode and the working electrode so as to be
`electrically insulated therefrom.
`The reaction layer can be formed over the working
`electrode and the counter electrode of the electrode system.
`Alternatively, the reaction layer can be formed over not only
`the working electrode and the counter electrode but also the
`third electrode.
`The reaction layer can be formed with one layer or two
`layers including at least an enzyme (oxidoreductase), and
`more preferably further including an electron acceptor.
`When the reaction layer is formed with two layers, the two
`layers can be a ?rst layer made of a hydrophilic polymer
`formed directly on the electrode system, and a second layer
`including at least an enzyme and an electron acceptor
`laminated on the ?rst layer.
`Examples of the hydrophilic polymer forming the ?rst
`hydrophilic polymer layer include carboxy methyl cellulose
`(hereinafter referred to as CMC), hydroxyethyl cellulose
`(hereinafter referred to as HEC), hydroxypropyl cellulose
`(hereinafter referred to as HPC), methyl cellulose, ethyl
`cellulose, ethyl hydroxyethyl cellulose, carboxymethyl ethyl
`cellulose, polyvinyl pyrrolidone, polyvinyl alcohol,
`polyamino acids such as polylysine, polystyrene sulfonate,
`gelatin or its derivative, acrylic acid or its salt, methacrylic
`acid or its salt, starch or its derivative, and maleic anhydride
`or its salt. Among the above, CMC, HEC, HPC, methyl
`cellulose, ethyl cellulose, ethyl hydroxyethyl cellulose and
`carboxymethyl ethyl cellulose are preferred.
`The kind of oxidoreductase contained in the reaction layer
`depends upon a substrate contained in a sample liquid to be
`quanti?ed and is not herein speci?cally de?ned. Examples
`of the oxidoreductase include fructose dehyrogenase, inver
`tase, mutarotase, glucose oxidase, alcohol oxidase, lactic
`acid oxidase, cholesterol oxidase, xanthine oxidase and
`amino acid oxidase.
`Examples of the electron acceptor include potassium
`ferricyanide, p-benzoquinone, phenazine methosulfate,
`methylene blue and a ferrocene derivative. One or a com
`bination of two or more of the above can be used as the
`electron acceptor.
`The enzyme and the electron acceptor can be dissolved in
`a sample liquid, or the reaction layer is immobilized on the
`substrate or the like so as not to allow the enzyme and the
`electron acceptor to dissolve in a sample liquid. When the
`enzyme and the electron acceptor are immobilized, the
`reaction layer preferably includes the hydrophilic polymer.
`The reaction layer can further include a pH buffer such as
`potassium dihydrogenphosphate-dipotassium hydrogen—
`phosphate,
`potassium
`dihydrogenphosphate-disodiurn
`hydrogenphosphate, sodium dihydrogenphosphate-dipotas
`sium hydrogenphosphate, sodium dihydrogenphosphate-di
`sodium hydrogenphosphate, citric acid-disodium hydrogen
`phosphate, citric acid-dipotassium hydrogenphosphate,
`citric acid-sodium citrate, citric acid-potassium citrate,
`potassium dihydrogencitrate-sodium hydroxide, sodium
`dihydrogencitrate-sodium hydroxide, sodium hydrogen
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`maleate-sodium hydroxide, potassium hydrogenphthalate
`sodium hydroxide, succinic acid-sodium tetraborate, maleic
`acid-tris(hydroxymethyl)aminomethane,
`tris(hydroxym
`ethyl)
`aminomethane-tris(hydroxymethyl)arninomethane
`hydrochloride, [N-(2-hydroxyethyl)piperazine-N'~2-ethane
`sulfonic acid]sodium hydroxide, [N-tris(hydroxymethyl)m
`ethyI-Z-aminoethanesulfonic acidl-sodium hydroxide, and
`[piperazine-N,N'-bis(2-ethanesulfonic
`acid)]-sodium
`hydroxide.
`The reaction layer can be formed by dropping on a
`substrate a solution including at least the enzyme and more
`preferably further including the electron acceptor and drying
`the dropped solution.
`When the reaction layer is formed with the ?rst hydro
`philic polymer layer and the second layer that includes the
`enzyme and the electron acceptor and is laminated, on the
`?rst layer, the second layer can be formed by, for example,
`dropping a mixed solution of the enzyme and the electron
`acceptor on the ?rst layer.
`Now, a method for quantifying a substrate in a sample
`liquid by using the biosensor will be described.
`The method can be carried out with a quantifying device
`comprising a measuring device A and the biosensor B
`removably connected to the measuring device A as is shown
`in FIG. 7 or 8.
`As is shown in FIGS. 7 and 8, the biosensor B comprises
`an electrode system formed on a substrate including a
`working electrode 5, a counter electrode 8 and a third
`electrode 7 used for detecting a liquid junction. The working
`electrode 5 is connected to a terminal 2a via a lead 2. The
`counter electrode 8 is connected to a terminal 4a via a lead
`4. The third electrode 7 is connected to a terminal 3a via a
`lead 3. The measuring device A shown in FIG. 7 comprises
`a connector 25 including terrrrinals 31, 32 and 33 connected
`to the respective terminals 2a, 3a and 4a of the biosensor B,
`current/voltage converting circuits 26 connected to the ter
`minal 32 of the connector 25, AID converting circuits 27
`connected to the respective current/voltage converting cir
`cuits 26, current/voltage converting circuits 26a connected
`to the terminal 31 via a switch 29, AID converting circuits
`27a connected to the respective current/voltage converting
`circuits 26a, and a controller 28 including a microcomputer
`and the like connected to the respective AID converting
`circuits 27 and 27a. The above-mentioned switch 29 is
`ON/OFF controlled by the controller 28.
`An operation of a circuit including the. biosensor B and
`the measuring device A of FIG. 7 will be described.
`First, the biosensor B is connected to the measuring
`device A. At this time, the switch 29 is disconnected from
`the terminal 31 by the controller 28. On the other hand, a
`predetermined voltage (e.g., 0.5 volts) is applied between
`the counter electrode 8 and the third electrode 7. When a
`sample liquid is supplied to the biosensor B under this
`condition, a current ?ows between the counter electrode 8
`and the third electrode 7. The value of this current is detected
`by the controller 28. Based on this detection, a time is
`measured. After a predetermined period of time, the switch
`29 is switched to the terminal 31 and a predetermined
`voltage (e.g., 0.5 volts) is applied to the working electrode
`5. A ?xed voltage (e.g., 0.5 volts) required for obtaining a
`response current is applied between the working electrode 5
`and the counter electrode 8 of the biosensor B. A current thus
`?owing between the working electrode 5 and the counter
`electrode 8 is converted into a voltage by the current/voltage
`converting circuits 26a, and the obtained voltage value is
`converted into the number of pulses in a ?xed period of time
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`by the AID converting circuits 27a. The controller 28 counts
`up the number of the pulses, calculates a response value, and
`indicates the result.
`Therefore, when a sample liquid including a substrate (for
`example, a saccharide) is supplied to the biosensor B, the
`reaction layer is dissolved in the sample liquid. After a
`predetermined period of time from the supply of the sample
`liquid, a ?xed voltage is applied between the working
`electrode 5 and the counter electrode 8. Then, after a
`predetermined period of time, a current value of a current
`?owing through the electrodes is measured. The obtained
`current value is in proportion to the concentration of the
`substrate in the sample liquid. A large number of current
`values are previously measured with regard to a plurality of
`sample liquids including the substrate at various known
`concentrations so as to obtain the relationship between the
`concentration and the current value. Then, the substrate
`contained in a sample liquid at an unknown concentration
`can be quanti?ed by measuring a current value as described
`above.
`In such a quantifying method in which the change of the
`concentration of a substrate caused through the reaction
`between an enzyme and the substrate in a sample liquid is
`measured based upon an electrochemical response obtained
`by applying a voltage to the working electrode 5, when the
`third electrode is used, as a reference electrode, in addition
`to the working electrode 5 and the counter electrode 8, the
`deviation of a reference potential can be substantially
`ignored.
`Furthermore, when the third electrode 7 is used for
`detecting a liquid junction, even though there is no need to
`apply a voltage between the working electrode 5 and the
`counter electrode 8 for the detection of the liquid junction as
`is conventionally applied, the supply of the sample liquid
`can be detected without fail. Ftuthermore, when the third
`electrode 7 is commonly used as a reference electrode and
`a liquid junction detecting electrode, the structure of the
`electrode system can be simpli?ed.
`The measuring device A shown in FIG. 8 comprises a
`connector 25 including terminals 31, 32 and 33 connected to
`the respective terminals 2a, 3a and 4a of the biosensor B,
`current/voltage converting circuits 26 connected to the
`respective terminals 31 or 32 of the connector 25 via a
`switch 29, AID converting circuits 27 connected to the
`respective current/voltage converting circuits 26, a control
`ler 28 including a microcomputer and the like connected to
`the respective AID converting circuits 27. The above~men
`tioned switch 29 is ON/OFF controlled by the controller 28.
`An operation of a circuit including the biosensor B and the
`measuring device A of FIG. 8 will be described.
`First, the biosensor B is connected to the measuring
`device A. At this time, the switch 29 is connected to the
`terminal 32 by the controller 28.. A predetermined voltage
`(e.g., 0.5 volts) is applied between the counter electrode 8
`and the third electrode 7. When a sample liquid is supplied
`to the biosensor B under this condition, a current ?ows
`between the counter electrode 8 and the third electrode 7.
`The value of this current is detected by the controller 28.
`Based on this detection, a time is measured. After a prede
`termined period of time, the switch 29 is switched to the
`terminal 31 and a predetermined voltage (e.g., 0.5 volts) is
`applied to the working electrode 5. A ?xed voltage (e.g., 0.5
`volts) required for obtaining a response current is applied
`between the working electrode 5 and the counter electrode
`8 of the biosensor B.
`A current thus ?owing between the working electrode 5
`and the counter electrode 8 is converted into a voltage by the
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`current/voltage converting circuits 26, and the obtained
`voltage value is converted into the number of pulses in a
`?xed period of time by the A/D converting circuits 27. The
`controller 28 counts up the number of the pulses, calculates
`a response value, and indicates the result.
`The measuring device A shown in FIG. 7 requires the
`current/voltage converting circuits 26 and 26a, and the A/D
`converting circuits 27 and 27a, respectively for the working
`electrode 5 and the counter electrode 8. In the measuring
`device A shown in FIG. 8, the current/voltage converting
`circuits 26 and the AID converting circuits 27 can be
`commonly used for the working electrode 5 and the counter
`electrode 8.
`The application of the biosensor of this invention depends
`upon a substrate (a speci?c component) in a sample liquid to
`be quanti?ed. The biosensor can be used as, for example, a
`fructose sensor, a sucrose sensor, a glucose sensor, an
`alcohol sensor, a lactic acid sensor, a cholesterol sensor and
`an amino acid sensor.
`
`EXAMPLES
`
`Throughout the drawings mentioned in the following
`description, the same el