Enzyme electrode and a biosensor and a measuring apparatus therewith

On an insulating substrate 1 is formed an electrode 2 as a working electrode, on which is formed a binding layer 3 mainly consisting of .gamma.-aminopropyltriethoxysilane. On the binding layer are sequentially formed an immobilized enzyme layer 4 in which a catalytic enzyme is immobilized in an organic polymer and a permeation restricting layer 5.

REFERENCE
 TO RELATED APPLICATION AND INCORPORATION BY REFERENCE This application is
 based on applications NO.HEI10-187528 filed in Japan and NO.HEI10-187529
 filed in Japan, the content of which is incorporated hereinto by
 reference.
 BACKGROUND OF THE INVENTION
 1. Field of the Invention
 This invention relates to an enzyme electrode for electrochemically
 determining a particular chemical substance in a solution via an enzyme
 reaction as well as a biosensor and a measuring apparatus therewith.
 2. Description of the Related Art
 A process employing an enzyme reaction in combination with an
 electrochemical reaction has been extensively used for determining a
 variety of components in, for example, a biological sample. For instance,
 there has been commonly used a biosensor in which by the catalytic action
 of an enzyme, a chemical compound in a solution is converted into hydrogen
 peroxide, which is then determined via an oxidation-reduction reaction.
 For example, a glucose biosensor can quantify an amount of glucose in a
 sample by oxidizing glucose with glucose oxidase (GOX) into gluconolactone
 and hydrogen peroxide, and determining the amount of the hydrogen peroxide
 because it is proportional to the glucose concentration.
 Such a type of sensor generally has a layer restricting permeation of a
 target chemical compound (hereinafter, referred to as a "permeation
 restricting layer") as the outermost layer in its electrode. FIG. 9 shows
 an enzyme electrode having such a structure where an electrode 2 as a
 working electrode is formed on an insulating substrate 1, on which there
 are sequentially formed a binding layer 3, an immobilized enzyme layer 4
 immobilizing a catalytic enzyme in an organic polymer and a permeation
 restricting layer 5. Such a permeation restricting layer may restrict an
 excessive diffusion of the target compound to make an upper limit in
 determination higher to some extent. In addition, it can prevent the
 immobilized enzyme layer from being in direct contact with a sample such
 as urine and blood which may cause deterioration in performance due to
 protein adhesion or decomposition of the enzyme. A permeation restricting
 layer has been made of, for example, polyalkylsiloxane (JP-A 10-26601) or
 silicone (JP-A 6-242068).
 JP-A 59-22620 has disclosed a biosensor comprising a permeation restricting
 layer having a different structure from the above in which a porous
 TEFLON.RTM. (tetrafluoroethylene) or polyfluorovinylidene film as a
 permeation restricting layer is placed, covering an electrode.
 U.S. Pat. No. 5,696,314 has disclosed an enzyme electrode in which a porous
 permeation restricting layer including TEFLON particles is formed on an
 immobilized enzyme layer. In the enzyme electrode, as shown in FIG. 11, an
 electrode 31 made of, for example, platinum and then an immobilized enzyme
 layer 32 are formed on a substrate 30. Then, a polymer layer 34 comprising
 the same enzyme as that in the immobilized enzyme layer 32 is formed via
 an adhesion layer 33, on which there are sequentially formed a permeation
 restricting layer 35, an adhesion layer 36 and a protection layer 37.
 The U.S. patent has disclosed a porous permeation restricting layer 35
 essentially comprising polymer particles, metal particles and polymer
 binder where the polymer particles and the polymer binder are made of
 TEFLON. The permeation-restriction layer 35 is formed by screen printing.
 Specifically, TEFLON binder is dissolved in a fluorine-containing solvent,
 particles such as alumina and TEFLON particles are added, and then the
 mixture is roll-milled into an ink. The prepared ink is stenciled to form
 the permeation restricting layer 35.
 The above prior art, however, has the following problems.
 There will be described problems in the case of using polyalkylsiloxane or
 silicone as a permeation restricting layer material. Such a material may
 cause a problem of insufficient durability for long-term use, which is due
 to inadequate strength of the permeation restricting layer. An enzyme
 electrode has a structure of plies made of organic materials such as an
 immobilized enzyme film which may be swollen in a solution. Therefore, the
 permeation restricting layer with inadequate strength may be intolerant to
 such film swelling, resulting in, for example, cracks. Thus, long-term use
 may cause failure of the enzyme electrode.
 When determination is repeated with a sample containing a higher level of
 contaminant for a long time, a sensor output may be remarkably reduced. It
 may be caused by deterioration of the original permeation restricting
 property due to adhesion of the contaminant to the permeation restricting
 layer. In particular, a body fluid may remarkably deteriorate the
 permeation restricting property because various materials such as urea
 compounds in addition to proteins are adhered to the permeation
 restricting layer.
 Furthermore, response may become slower when extending a concentration
 range to be measured to a higher level because when a sample containing a
 higher level of target compound is analyzed using a conventional
 permeation restricting layer, it is inevitable to increase the film
 thickness due to its limitation in selective permeability, leading to a
 longer time for stabilizing a diffusion rate within the permeation
 restricting layer.
 A technique using a TEFLON or polyfluorovinylidene (JP-A 59-22620) has the
 following problem.
 A technique using a filter comprising, for example, TEFLON has been
 conventionally used, where the filter is usually disposed outside of an
 enzyme electrode, covering the electrode because the fluorine compound is,
 as apparent from its molecular structure, less adhesive to other organic
 polymer layers such as an immobilized enzyme layer and thus the filter
 cannot be formed together with the layers including the immobilized enzyme
 layer. JP-A 59-22620 has disclosed only a configuration where a film
 consisting of the above fluorine compound is formed on the tip of the
 enzyme electrode, but not a configuration where the film is adhesively
 formed in the electrode surface.
 Thus, the above prior art has problems that there is formed a certain gap
 between the permeation restricting layer and the electrode surface,
 resulting that 1) response becomes slower due to a longer time for a
 target compound to reach the electrode surface and 2) it takes a longer
 time for washing, which leads to a longer waiting time for the next
 determination.
 A permeation restricting layer comprising the above fluorine compound must
 have pores with a diameter of 10 to 100 .mu.m and be thick adequately to
 be permeation-restrictive. Thus, response becomes slower and it takes a
 longer time for washing, leading to a longer waiting time for the next
 determination.
 Furthermore, a permeation restricting layer comprising the above fluorine
 compound is less flexible. Therefore, the structure is readily broken when
 a layer disposed nearer to the electrode than the permeation restricting
 layer is swollen. In particular, the problem is significant when the
 permeation restricting layer is adjacent to an expansive immobilized
 enzyme layer.
 On the other hand, U.S. Pat. No. 5,696,314 has disclosed a configuration
 where a permeation restricting layer comprising TEFLON particles and
 TEFLON binder is formed on an electrode as one part.
 As described above, a permeation restricting layer of a polymer with a
 higher fluorine content such as TEFLON is less adhesive to an adjacent
 polymer layer such as an immobilized enzyme layer. Therefore, even when
 the permeation restricting layer is formed with, for example, an
 immobilized enzyme layer as one part, adhesive strength is insufficient in
 an interface between these layers. Furthermore, since a permeation
 restricting layer comprising TEFLON is less flexible, it cannot follow a
 swollen adjacent layer. Thus, there may readily occur detachment between
 the permeation restricting layer and its adjacent layer such as an
 immobilized enzyme layer during operation. Once detachment occurs, there
 is formed a certain gap between the permeation restricting layer and the
 electrode surface, resulting that 1) response becomes slower due to a
 longer time for a target compound to reach the electrode surface and 2) it
 takes a longer time for washing, which leads to a longer waiting time for
 the next determination.
 When using TEFLON as described in the above publication, it is difficult to
 prepare a solution due to its less solubility to a solvent. It is,
 therefore, difficult to deposit a layer by a general process such as spin
 coating and thus to make the permeation restricting layer thinner.
 Furthermore, a permeation restricting layer comprising the above fluorine
 compound must be porous for its permeation restricting property, and
 therefore, must be thick to some extent. According to the U.S. patent, the
 thickness is preferably 10 to 40 .mu.m. Thus, it is inevitable to make the
 permeation restricting layer thick, leading to slower response and a
 longer washing time.
 In addition, a permeation restricting layer comprising TEFLON is less
 flexible as described above, and therefore tends to be broken when an
 adjacent layer is swollen. The problem should be also improved. The
 problem is particularly significant when the permeation-restriction layer
 is adjacent to an expansive immobilized enzyme layer.
 Another technique of the prior art using a fluorine compound will be
 described, which is not related to an application as a component for a
 permeation restricting layer.
 A fluorine-compound film (TEFLON film) 10 to 50 .mu.m of thickness has been
 commonly used as an oxygen permeable film and described in, for example,
 JP-A 56-73342. The film is, however, generally disposed between an
 immobilized enzyme layer and an electrode, but not on the immobilized
 enzyme layer. It, therefore, does not act as a permeation restricting
 layer.
 It is also well-known that a NAFION.RTM. film, an ion- exchange film, is
 disposed on an immobilized enzyme layer, which has been disclosed in, for
 example, JP-A 8-50112. NAFION is a cation-exchange polymer in which
 perfluoroalkylene ether side chains having a terminal sulfonic group are
 attached to a perfluoromethylene principal chain (Formula 1).
 ##STR1##
 A NAFION film disposed on the immobilized enzyme layer may minimize
 back-diffusion of hydrogen peroxide, reduce variation with time of
 response to glucose after reaching a peak value and improve response
 properties. The film is, however, not adequately effective as a permeation
 restricting layer due to its terminal sulfonic groups. An ion-exchange
 film is used for preventing permeation of ionic interferent materials
 interfering an electrode reaction, but little restricts permeation of, for
 example, excessive glucose.
 For a biosensor, it is important to eliminate effects of interferent
 materials or contaminants. An interferent material refers to a chemical
 substance which may affect the above oxidation-reduction reaction system
 to give a positive error in a measurement result, such as ascorbic acid
 and acetaminophen. A contaminant refers to a chemical substance which may
 be adsorbed by an electrode surface to give a negative error in a
 measurement result. For example, Bioindustry, Vol.9, No.12, pp.20-25
 (1992) has list albumin, urea, urea compounds and creatinine as a
 contaminant to a sensor output, i.e., a substance giving a negative error.
 JP-A 8-180286 has disclosed a biosensor in which a permeation restricting
 layer of polyalkylsiloxane and NAFION or acetylcellulose is deposited as
 an enzyme electrode for eliminating effects of a higher level of
 interferent materials on a sensor output. FIG. 26 shows its configuration
 where an electrode 12 is formed on an insulating substrate 11, on which
 there are sequentially formed a y-aminopropyl-triethoxysilane film 13, an
 acetylcellulose film 14, a perfluorocarbonsulfonic acid film 15, an
 immobilized enzyme layer 16 and a polyalkylsiloxane film 17. The
 publication describes that such a layered structure may prevent a higher
 level of interferent materials from reaching an electrode surface.
 JP-A 3-72254 has disclosed a biosensor in which a permeation restricting
 layer of NAFION and polyurethane is deposited as an enzyme electrode for
 eliminating effects of interferent materials or contaminants on a sensor
 output. FIG. 27 shows its configuration where a working electrode 23, a
 control electrode 24 and an insulative protection film 25 are formed on a
 plastic film 22 and an immobilized enzyme layer 29 is formed covering
 these films. The immobilized enzyme layer 29 consists of a NAFION film
 29a, an enzyme layer 29b and a polyurethane layer 29c. The publication
 describes that such a structure of electrode may reduce permeation or
 adhesion of an interferent or concomitant material to an electrode.
 It has been difficult to eliminate a contaminant such as urea compounds
 during measurement for a body fluid such as urine or blood containing the
 contaminant at a higher level when such an enzyme electrode is used. For
 example, an acetylcellulose film can restrict permeation of a higher
 molecular-weight compound, but not adequately restrict permeation of a
 lower molecular-weight compound such as urea.
 Therefore, a contaminant such as urea compounds may reach an electrode
 surface to be irreversibly adsorbed. Thus, repeated use may cause
 reduction in a sensor output with time, and resultantly such a sensor is
 less reproductive for repeated measurement or less stable for a long-term
 use. Furthermore, an urea compound, once adsorbed by an electrode, cannot
 be easily removed by washing with water. It may lead to a longer waiting
 time for the next measurement and particularly for repeated measurement,
 an accumulated negative error may be more significant depending on the
 measurement number.
 Such a problem of adsorption of a contaminant such as an urea compound is
 significant particularly when platinum is used as an electrode material
 because an urea compound tends to adhere to platinum. However, an
 electrode is generally made of platinum having good chemical resistance
 and good detection properties for hydrogen peroxide. Thus, it has been
 strongly desired to develop an enzyme electrode which may solve the above
 problems.
 SUMMARY OF THE INVENTION
 In the light of the above problems, an objective of this invention is to
 provide an enzyme electrode allowing highly sensitive and stable
 measurement even in a long-term use as well as a biosensor and a measuring
 apparatus therewith.
 For achieving the objective, this invention provides an enzyme electrode
 which can perform detection under a wide range of working conditions and
 has a good durability for a long-term use, by forming a permeation
 restricting layer with a particular structure of polymer.
 Furthermore, this invention can eliminate effects of contaminants or
 interferent materials on a measured value in determination for a body
 fluid sample such as urine, blood and sweat containing urea compounds at a
 higher level and prevent reduction of a sensor output for a consistently
 reproductive output, by forming an electrode protection layer comprising
 an urea compound.
 This invention will be described in detail.
 This invention provides an enzyme electrode comprising an electrode on an
 insulating substrate, an immobilized enzyme layer on the electrode and a
 permeation restricting layer on the immobilized enzyme layer, said
 permeation restricting layer mainly consisting of a polymer in which a
 pendant group containing at least a fluoroalkylene block is attached to a
 non-fluorinated vinyl polymer.
 The enzyme electrode consists of an electrode (electrode layer) on an
 insulating substrate and multiple layers having different functions on the
 electrode. The permeation restricting layer is made of a polymer having a
 pendant group comprising a fluoroalkylene block (fluoroalkylene unit).
 Such a configuration may prevent adhesion of contaminants such as proteins
 and urea compounds to provide an enzyme electrode exhibiting stable output
 properties even for a long-term use. The fluoroalkylene moiety may not
 contribute to dissolution into a washing agent such as non-fluorinated
 solvent and a surfactant, providing an enzyme electrode with good chemical
 resistance.
 The polymer has a non-fluorinated vinyl polymer structure as a principal
 chain, which is highly adhesive to another organic polymer layer such as
 an immobilized enzyme layer. It, therefore, does not cause a gap between a
 layer such as the immobilized enzyme layer on the electrode surface and
 the permeation restricting layer. It may allow response to be faster and a
 time for washing to be reduced. In addition, its good adhesiveness may
 improve durability of the layered structure to provide an enzyme electrode
 resistant to deterioration due to a long-term use. The polymer may have,
 in addition to the pendant group comprising a fluoroalkylene block, any
 other appropriate side chain or functional group; for example, a properly
 polar functional group such as --OH and --COOH groups may further improve
 adhesiveness to another organic polymer layer such as an adjacent
 immobilized enzyme layer.
 The polymer composing the permeation restricting layer has a unique
 structure that a pendant group comprising at least a fluoroalkylene block
 is attached to a non-fluorinated vinyl polymer chain, exhibiting a good
 permeation restricting property when used in, for example, a glucose
 sensor. Thus, it may significantly extend a measurable concentration
 range. Furthermore, its excellent permeation restricting property may
 allow the permeation restricting layer to be thinner, for example, below
 0.1 .mu.m, leading to faster response and a reduced time for washing.
 The permeation restricting layer may be formed as a homogeneous film by a
 convenient process such as dip-coating, spin-coating and spray-coating and
 may be suitable for mass production.
 This invention also provides an enzyme electrode comprising an electrode on
 an insulating substrate, an immobilized enzyme layer on the electrode and
 a permeation restricting layer on the immobilized enzyme layer, said
 permeation restricting layer consisting of a polycarboxylic acid (A)
 fluoroalcohol ester.
 This invention also provides an enzyme electrode comprising an electrode on
 an insulating substrate, an immobilized enzyme layer on the electrode and
 a permeation restricting layer on the immobilized enzyme layer, said
 permeation restricting layer comprising a polycarboxylic acid (A)
 fluoroalcohol ester and a polycarboxylic acid (B) alkylalcohol ester.
 This invention also provides an enzyme electrode comprising an electrode on
 an insulating substrate, an immobilized enzyme layer on the electrode and
 a permeation restricting layer on the immobilized enzyme layer, said
 permeation restricting layer mainly consisting of a polycarboxylate
 comprising alkylalcohol ester and fluoroalcohol ester groups.
 These enzyme electrodes consist of an electrode (electrode layer) on an
 insulating substrate and multiple layers having different functions on the
 electrode, characterized in that the permeation restricting layer is
 composed of a polymer having a particular structure.
 An enzyme electrode according to this invention is made of a fluoroalcohol
 polycarboxylate. A fluoroalcohol polycarboxylate refers to a
 polycarboxylic acid, whose carboxyl groups are partially or totally
 esterified with a fluoroalcohol. A fluoroalcohol refers to an alcohol, at
 least one or all of whose hydrogen atoms are replaced with fluorine
 atom(s).
 The permeation restricting layer material has a fluoroalcohol ester group,
 which may prevent adhesion of contaminants such as proteins and urea
 compounds, leading to an enzyme electrode exhibiting a stable output
 property when used for a long term.
 The permeation restricting layer material having a fluoroalcohol ester
 group may prevent adhesion of contaminants such as proteins and urea
 compounds, leading to an enzyme electrode exhibiting a stable output
 property for a long-term use. The fluoroalcohol ester group may not
 contribute to dissolution into a washing agent such as almost all kinds of
 non-fluorinated solvents and surfactants, providing an enzyme electrode
 with good chemical resistance.
 These enzyme electrodes have a permeation restricting layer comprising a
 polymer having a principal chain of a polycarboxylic acid, to which a
 fluoroalcohol is attached via an ester group. The polymer is highly
 adhesive to another organic polymer layer such as an immobilized enzyme
 layer. It, therefore, does not cause a gap between a layer such as the
 immobilized enzyme layer on the electrode surface and the permeation
 restricting layer. It may allow response to be faster and a time for
 washing to be reduced. In addition, its good adhesiveness may improve
 durability of the layered structure to provide an enzyme electrode
 resistant to deterioration due to a long-term use. The polymer may have,
 in addition to the fluoroalcohol ester group, any other appropriate
 functional group to the principal chain; a properly polar functional group
 may further improve adhesiveness to another organic polymer layer such as
 an adjacent immobilized enzyme layer.
 The polymer composing the permeation restricting layer has a unique
 structure that the carboxyl groups of the polycarboxylic acid are
 partially or totally esterified with a fluoroalcohol, which may
 significantly extend a measurable concentration range when used in, for
 example, a glucose sensor. Furthermore, its excellent permeation
 restricting property may allow the permeation restricting layer to be
 thinner, for example, below 0.1 .mu.m, leading to faster response and a
 reduced time for washing.
 The permeation restricting layer may be formed as a homogeneous film by a
 convenient process such as dip-coating, spin-coating and spray-coating and
 may be suitable for mass production.
 When a permeation restricting layer comprises (a) a polycarboxylic acid (A)
 fluoroalcohol ester and a polycarboxylic acid (B) alklylalcohol ester or
 (b) a polycarboxylate having an alkylalcohol ester and a fluoroalcohol
 ester groups, high temperature stability may be improved, in addition to
 the above effects. An enzyme electrode or a biosensor therewith may be
 sometimes stored or used at a relatively higher temperature (for example,
 ca. 40.degree. C.). When used for measurement after leaving at a higher
 temperature, sensitivity of a conventional enzyme electrode has often
 varied significantly, compared to measurement before exposure to the
 higher temperature. On the other hand, an enzyme electrode or biosensor
 comprising the above permeation restricting layer little varies in its
 sensitivity even after exposure to a higher temperature.
 In an enzyme electrode of this invention as described above, an electrode
 and an immobilized enzyme layer may be formed in direct contact with each
 other or another layer may intervene these layers. For example, a binding
 layer mainly consisting of a silane coupling agent is disposed between the
 electrode and the immobilized enzyme layer or an ion-exchange polymer
 layer mainly consisting of an ion- exchange polymer having a
 perfluorocarbon skeleton is disposed between the binding layer and the
 immobilized enzyme layer. Similarly, the immobilized enzyme layer and the
 permeation restricting layer may be formed in direct contact with each
 other or another layer may intervene these layers.
 This invention also provides a method for manufacturing an enzyme electrode
 comprising the steps of: forming an electrode on an insulating substrate,
 applying the first liquid containing an enzyme to the electrode directly
 or via another layer and then drying it to form an immobilized enzyme
 layer, and applying the second liquid containing a polymer in which a
 pendant group having at least a fluoroalkylene block is attached to a
 non-fluorinated vinyl polymer, to the immobilized enzyme layer directly or
 via another layer and then drying it to form a permeation restricting
 layer.
 In this manufacturing process, a permeation restricting layer is formed by
 applying and then drying the second liquid comprising a polymer having the
 above particular structure. Thus, there may be provided, with a good
 controllability for a film thickness, a permeation restricting layer which
 is excellent in stability for repeated measurement, adhesiveness to
 adjacent layers, durability and permeation restricting property. Since the
 second liquid has a lower viscosity, the permeation restricting layer may
 be readily formed with a reduced film thickness. Specifically, a
 permeation restricting layer 0.01 to 3 .mu.m of thickness after drying may
 be satisfactorily formed.
 This invention also provides a biosensor using the above enzyme electrode
 as a working electrode. The biosensor has a permeation restricting layer
 comprising the polymer having the above particular structure on the enzyme
 electrode surface. It, therefore, may be excellent in long-term stability
 and may be used under a wide range of measuring conditions.
 Although the above description is related to an enzyme electrode and other
 entities comprising a permeation restricting layer composed of a polymer
 having a particular structure, this invention also provides an enzyme
 electrode and other entities comprising an electrode protection layer
 having a particular structure.
 This invention also provides an enzyme electrode comprising an electrode on
 an insulating substrate, an electrode protection layer comprising an urea
 compound covering at least a part of the electrode, and an immobilized
 enzyme layer covering the electrode and the electrode protection layer.
 The enzyme electrode consists of an electrode (electrode layer) on an
 insulating substrate and multiple layers having different functions on the
 electrode, which may be used as a detection element in a biosensor, in
 particular a biosensor for determining, for example, glucose in a sample
 such as urine, blood and sweat.
 In this enzyme electrode, an electrode protection layer containing an urea
 compound is formed on an electrode surface on an insulating layer. An urea
 compound is, as described above, a contaminant which may give a negative
 error to a measurement result when adhering to the electrode surface. The
 enzyme electrode of this invention has an electrode protection layer
 comprising such a contaminant on the electrode surface in advance, for
 preventing contaminants in a sample from reaching the electrode surface
 during measurement and thus minimizing variation in sensitivity.
 An electrode protection layer comprising an urea compound may degrade
 absolute sensitivity, but the degree may be practically insignificant. On
 the other hand, sensitivity variation with time associated with repeated
 operation may be remarkably improved in comparison with a conventional
 type of enzyme electrode which does not have an electrode protection
 layer.
 Furthermore, an electrode protection layer may restrict permeation of
 interferent materials such as ascorbic acid and acetaminophen. Compared
 with permeation of these interferent materials, the layer may be less
 restrictive to permeation of hydrogen peroxide. Thus, the layer may
 improve selective permeability for hydrogen peroxide.
 The enzyme electrode of this invention has an electrode protection layer
 functioning as described above. Thus, a sensor output reduction with time
 due to an urea compound and a sensor output increase due to interferent
 materials are prevented for a stable output. Furthermore, it may provide a
 more sensitive sensor than that according to the prior art.
 This invention also provides a method for manufacturing an enzyme electrode
 comprising the steps of: forming an electrode on an insulating substrate
 surface, and then applying electricity to the insulating substrate while
 being soaked in a mixed solution comprising a supporting electrolyte and
 an urea compound to cover at least a part of the electrode with an
 electrode protection layer comprising the urea compound.
 The process for manufacturing an enzyme electrode employs electrolysis for
 selectively forming urea-compound layers on individual electrodes. Forming
 an urea-compound layer and patterning may be simultaneously conducted,
 which may simplify the manufacturing process and readily lead to mass
 production. Furthermore, the urea-compound layer may be formed,
 independently of the size or shape of the electrode.
 This invention also provides a biosensor using the above enzyme electrode
 as a working electrode. The biosensor has an enzyme electrode having an
 electrode protection layer comprising an urea compound which covers at
 least a part of the electrode. It, therefore, may prevent sensor output
 reduction with time due to urea compounds and sensor output increase due
 to interferent materials for giving a stable output. Furthermore, it may
 realize higher sensitivity than that of the prior art. A biosensor
 according to this invention may have further have a counter electrode and
 a reference electrode on an insulating substrate. It is preferable that
 the enzyme electrode (working electrode) and the counter electrode are
 made of platinum while the reference electrode is made of silver/silver
 chloride because urea is easily attached to platinum so that an electrode
 protection layer comprising an urea compound may be suitably formed on the
 enzyme electrode (working electrode) or the counter electrode.
 This invention also relates to a variety of measuring apparatus using the
 above biosensor. Specifically, this invention provides a measuring
 apparatus comprising the above biosensor and a data indicator indicating
 an electric signal from the biosensor.
 This invention also provides a measuring apparatus comprising the above
 biosensor, an electrochemical measuring circuit receiving an electric
 signal from the biosensor, a data processor calculating a measured value
 based on the electric signal and a data indicator indicating the measured
 value.
 These measuring apparatus may realize highly sensitive and stable
 measurement because of their biosensor having a particular structure of
 working electrode. They are also easily operated even by an unfamiliar
 individual.
 As described above, an enzyme electrode of this invention comprising a
 permeation restricting layer mainly consisting of a particular structure
 of polymer or a biosensor therewith has the following advantages.
 First, it may prevent adhesion of contaminants such as proteins and urea
 compounds to realize a stable output property for a long-term use because
 a pendant group containing a fluoroalkylene block such as a fluoroalcohol
 ester group contributes to insolubility in a washing agent such as almost
 all non-fluorinated solvents and surfactants. It, therefore, may give
 stable repeated measuring results even for a test system comprising a
 variety of chemical compounds such as a body fluid.
 Secondly, the permeation restricting layer has good adhesiveness to another
 organic polymer layer such as an immobilized enzyme layer, leading to
 faster response, a reduced time for washing and improved durability of the
 layered structure, to provide an enzyme electrode unsusceptible to damage
 due to a long-term use. Such good adhesiveness may be endowed because the
 polymer has a principal chain consisting of a non-fluorinated vinyl
 polymer. A structure in which a pendant group is attached to a principal
 chain via an ester group may further improve adhesiveness.
 Thirdly, it may provide a good permeation-restricting property to
 significantly extend a measurable concentration range. Such a good
 permeation-restricting property may be endowed because the polymer
 composing of the permeation restricting layer has a particular structure
 in which a pendant group containing at least a fluoroalkylene block is
 attached to a non-fluorinated vinyl polymer chain.
 Fourthly, a good permeation-restricting property may allow the permeation
 restricting layer to be thinner for achieving faster response and a
 reduced time for washing.
 Fifthly, it may allow stable measurement for an ionized substance such as
 lactic acid because the permeation restricting layer has no charges and
 therefore little interacts with an ionic substance.
 An enzyme electrode of this invention comprising an electrode protection
 layer containing an urea compound and a biosensor therewith may be used to
 precisely determine a particular ingredient in a sample containing a
 higher level of contaminants such as urea because the electrode protection
 layer prevents the contaminants such as urea in the sample from permeating
 to the electrode. The electrode protection layer can prevent permeation of
 interferent materials such as ascorbic acid and acetaminophen, to improve
 selective permeation of a target substance such as hydrogen peroxide.
 Thus, the enzyme electrode or the biosensor of this invention may prevent
 sensor output reduction with time due to urea compounds and sensor output
 increase due to interferent materials for a stable output. Furthermore,
 such improvement in selectivity may realize a higher sensitive sensor than
 that of the prior art.
 An electrode protection layer containing an urea compound may be formed by
 electrolysis. Thus, urea- compound layers may be selectively formed on
 individual electrodes. Therefore, forming an urea-compound layer and
 patterning may be simultaneously conducted, which may simplify the
 manufacturing process and readily lead to mass production. Furthermore,
 the urea-compound layer may be formed, independently of the size or shape
 of the electrode.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
 As used herein, an "enzyme electrode" refers to an electrode comprising an
 immobilized enzyme layer. A "biosensor" refers to a device element
 comprising the enzyme electrode and, as necessary, a counter electrode
 and/or a reference electrode. As used herein, a "measuring apparatus"
 refers to a system comprising the above biosensor and equipped with a
 variety of means for indicating and processing an electric signal from the
 biosensor. An enzyme electrode, a biosensor and a measuring apparatus
 according to this invention will be described in detail.
 The first enzyme electrode according to this invention comprises an
 electrode on an insulating substrate, an immobilized enzyme layer on the
 electrode and a permeation restricting layer on the immobilized enzyme
 layer where the permeation restricting layer mainly consists of a polymer
 in which a pendant group containing at least a fluoroalkylene block is
 attached to a non-fluorinated vinyl polymer.
 The term "mainly consist(ing) of" means that the above polymer is a main
 component composing of the permeation restricting layer; for example, the
 polymer is contained in a proportion of 50 wt % or higher in the
 permeation restricting layer.
 A "non-fluorinated vinyl polymer" is a moiety for improving adhesiveness to
 another organic polymer layer such as an immobilized enzyme layer. There
 are, therefore, no limitations in terms of its structure, but it must have
 no fluorine atoms. If a polymer part other than a pendant group contains a
 fluorine atom, a permeation restricting layer may be less adhesive to
 another organic polymer layer such as an immobilized enzyme layer, making
 it difficult to prepare a solution or to form a permeation restricting
 layer as a thin film.
 A non-fluorinated vinyl polymer is a polymer having a principal chain
 composed of carbon-carbon chain; preferably a homopolymer or copolymer of
 one or more monomers selected from the group consisting of unsaturated
 hydrocarbons, unsaturated carboxylic acids and unsaturated alcohols; most
 preferably a polycarboxylic acid. An appropriate polymer may be selected
 for improving adhesiveness to another organic polymer layer such as an
 immobilized enzyme layer to provide a permeation restricting layer having
 good durability. It is preferable that a fluoroalkylene block is attached
 to a vinyl polymer via an ester group, which is appropriately polar to
 further improve adhesiveness to another organic polymer layer such as an
 immobilized enzyme layer. Such polymers include 1H,1H-perfluorooctyl
 polymethacrylate and 1H,1H,2H,2H-perfluorodecyl polyacrylate.
 A pendant group containing a fluoroalkylene block is one having a
 fluoroalkylene group as a unit. A fluoroalkylene group means an alkylene
 group whose hydrogens are partially or totally replaced with fluorines. A
 fluorine content in the pendant group, i.e., a value of x/(x+y) where x
 and y are the numbers of fluorine and hydrogen atoms in the pendant group,
 respectively, is preferably 0.3 to 1, more preferably 0.8 to 1, which may
 prevent adhesion of contaminants to the permeation restricting layer and
 give good permeation-restricting property.
 The pendant group preferably has 3 to 15 carbon atoms, more preferably 5 to
 10 carbon atoms, most preferably 8 to 10 carbon atoms, to make the length
 of the pendant group appropriate for providing good film-deposition
 property and permeation-restricting property and maintaining good
 adhesiveness to an adjacent polymer layer.
 The binding rate of the pendant group to the vinyl polymer, i.e., the
 content of the pendant group, is not particularly limited, but may be
 appropriately determined depending on the other polymers and its
 application; for example, 0.1 to 30%. The content of the water-repellent
 pendant group may be thus selected in an appropriate range to realize good
 permeation-restriction property and good adhesiveness to an adjacent
 polymer layer. The binding rate of the pendant group means the proportion
 of the pendant group to all the groups attached to the carbon--carbon
 chain as the principal chain of the vinyl polymer. For example, where a
 vinyl polymer as a principal chain is polyacrylate, 10% of whose --COOH
 groups are esterified to be a pendant group, the binding rate of the
 pendant group is 2.5% obtained by multiplying the binding rate of --COOH
 group, 25%, by the esterification rate, 10%.
 The polymer composing of the permeation restricting layer may be preferably
 1000 to 50000, more preferably 3000 to 20000. If it is too high, it is
 difficult to prepare a solution, while if it is too low, adequate
 permeation-restricting property is not provided. A molecular weight herein
 is a number average molecular weight.
 The second enzyme electrode according to this invention comprises an
 electrode on an insulating substrate, an immobilized enzyme layer on the
 electrode and a permeation restricting layer on the immobilized enzyme
 layer where the permeation restricting layer mainly consists of a
 polycarboxylic acid (A) fluoroalcohol ester. The term "mainly consist(ing)
 of" means that the above polymer is a main component composing of the
 permeation restricting layer; for example, the polymer is contained in a
 proportion of 50 wt % or higher in the permeation restricting layer.
 The third enzyme electrode according to this invention comprises an
 electrode on an insulating substrate, an immobilized enzyme layer on the
 electrode and a permeation restricting layer on the immobilized enzyme
 layer where the permeation restricting layer comprises a polycarboxylic
 acid (A) fluoroalcohol ester and a polycarboxylic acid (B) alkylalcohol
 ester.
 The fourth enzyme electrode according to this invention comprises an
 electrode on an insulating substrate, an immobilized enzyme layer on the
 electrode and a permeation restricting layer on the immobilized enzyme
 layer where the permeation restricting layer mainly consists of a
 polycarboxylate having an alkylalcohol ester and a fluoroalcohol ester
 groups. The term "mainly consist(ing) of" means that the above polymer is
 a main component composing of the permeation restricting layer; for
 example, the polymer is contained in a proportion of 50 wt % or higher in
 the permeation restricting layer.
 Examples of the polycarboxylic acid composing of the polycarboxylic acid
 (A) or (B) or the above polycarboxylate include polymers having a
 carboxylic acid unit such as acrylic acid, methacrylic acid, fumaric acid
 and itaconic acid; specifically polymethacrylic acid, polyacrylic acid and
 a copolymer of acrylic acid and methacrylic acid. The polycarboxylic acids
 (A) and (B) may be the same or different.
 A fluorine content in the fluroalcohol ester group, i.e., a value of
 x/(x+y) where x and y are the numbers of fluorine and hydrogen atoms in
 the fluoroalcohol ester group, respectively, is preferably 0.3 to 1, more
 preferably 0.8 to 1, which may prevent adhesion of contaminants to the
 permeation restricting layer and give good permeation-restricting
 property.
 The fluoroalcohol moiety composing of the fluoroalcohol ester group
 preferably has 3 to 15 carbon atoms, more preferably 5 to 10 carbon atoms,
 most preferably 8 to 10 carbon atoms, to make the length of the
 fluoroalcohol ester group appropriate for providing good film-deposition
 property and permeation-restricting property and maintaining good
 adhesiveness to an adjacent polymer layer.
 The esterification rate of the polycarboxylic acid fluoroalcohol ester is
 not particularly limited, but may be appropriately determined depending on
 the other polymers and its application; for example, 0.1 to 30%. The
 esterification rate means the rate of the esterified carboxylic acid
 groups belonging to the polyacrylic acid moiety in the principal chain.
 The esterification rate may be selected within the above range to make the
 content of the water-repelling fluoroalcohol ester group proper for
 realizing good permeation-restriction property and good adhesiveness to an
 adjacent polymer layer.
 In this invention, the fluoroalcohol composing of the fluoroalcohol ester
 is preferably a primary alcohol because it may effectively prevent
 adhesion of contaminants to the permeation restricting layer and may
 provide excellent chemical resistance to acids, alkalis or a variety of
 organic solvents. Preferable examples of the alcohol include
 1H,1H-perfluorooctyl polymethacrylate and 1H,1H,2H,2H-perfluorodecyl
 polyacrylate.
 The permeation restricting layer may comprise a polycarboxylic acid (A)
 fluoroalcohol ester and a polycarboxylic acid (B) alkylalcohol ester or
 may mainly consist of a polycarboxylate comprising an alkylalcohol ester
 and a fluoroalcohol ester groups, to provide an enzyme electrode
 exhibiting improved high-temperature stability.
 A preferable type of "fluoroalcohol ester" is as described above.
 The alkylalcohol moiety in the alkylalcohol ester part means a straight or
 circular alcohol represented by C.sub.n H.sub.n+2 OH (n is a natural
 number) where n is an integer of 1 or more, preferably 2 to 10, more
 preferably 4 to 8, most preferably 6. For example, hexyl and cyclohexyl
 groups may be suitable. Thus, stability of the enzyme electrode may be
 further improved when exposed to an elevated temperature.
 When the permeation restricting layer comprises a polycarboxylic acid (A)
 fluoroalcohol ester and a polycarboxylic acid (B) alkylalcohol ester, the
 content of the polylcarboxylic acid (A) fluoroalcohol ester to the overall
 permeation restricting layer is preferably 50 to 99 wt %, more preferably
 75 to 99 wt %, most preferably 80 to 95 wt %. If the content is too low,
 the permeation restricting layer may be less durable while if the content
 is too high, the permeation restricting layer may exhibit insufficient
 stability when exposed to an elevated temperature. On the other hand, the
 content of the polylcarboxylic acid (B) alkylalcohol ester to the overall
 permeation restricting layer is preferably 1 to 50 wt %, more preferably 1
 to 25 wt %, most preferably 5 to 20 wt %. If the content is too low, the
 permeation restricting layer may exhibit insufficient stability when
 exposed to an elevated temperature, while if the content is too high, the
 permeation restricting layer may be less durable. A polycarboxylic acid
 (B) alkylalcohol ester means a polycarboxylic acid (B) which is at least
 partially esterified with the above alkylalcohol; preferably cyclohexyl
 polymethacrylate.
 When the permeation restricting layer comprises a polycarboxylate
 comprising an alkylalcohol ester and a fluoroalcohol ester groups,
 preferable types of individual ester groups are as described above and a
 variety of combination of these ester groups may be employed. The ratio
 between the alkylalcohol ester and the fluoroalcohol ester groups is not
 particularly limited, but a/b where "a" and "b" are the numbers of the
 fluoroalcohol ester group and of the alkylalcohol ester group,
 respectively, is preferably 50/50 to 99/1, more preferably 75/25 to 99/1,
 most preferably 80/20 to 95/5.
 Preferable polycarboxylates include those containing a cyclohexyl
 polymethacrylate unit; for example those containing the repeating unit
 represented formula (2), which may improve high-temperature stability and
 permeation-restricting property.
 ##STR2##
 where n is an integer of 2 or more, X is an integer of 0 or more, and Y is
 an integer of 1 or more.
 Specific compounds include a copolymer of 1H,1H-perfluorooctyl methacrylate
 and cyclohexyl methacrylate and a copolymer of lH,lH,2H,2H-perfluorodecyl
 acrylate and cyclohexyl methacrylate; preferably a compound represented by
 formula (3) having the repeating units of 1H,1H,2H,2H-perfluorodecyl
 acrylate and of cyclohexyl methacrylate.
 ##STR3##
 where n is an integer of 2 or more.
 These copolymers may be used to improve particularly high-temperature
 stability and other properties such as permeation-restricting property.
 A permeat ion restricting layer in an enzyme electrode according to this
 invention comprises a particular structure of polymer, but may comprises a
 mixture of two or more polymers whose structures and/or molecular weights
 are different from each other.
 For the above first to fourth enzyme electrodes, the molecular weight of a
 polylcarboxylic acid (A) fluoroalcohol ester or a polycarboxylate
 containing an alkylalcohol ester and a fluoroalcohol ester groups
 composing of the permeation restricting layer is preferably 1000 to 50000,
 more preferably 3000 to 30000. If it is too high, it is difficult to
 prepare a solution, while if it is too low, adequate
 permeation-restricting property is not provided. A molecular weight herein
 is a number average molecular weight.
 In these enzyme electrodes, the thickness of the permeation restricting
 layer is preferably 0.01 to 3 .mu.m, more preferably 0.01 to 1 .mu.m, most
 preferably 0.01 to 0.1 .mu.m, which may lead to improvement in response
 speed and reduction in a washing time.
 This invention also provides a method for manufacturing an enzyme electrode
 comprising the steps of: forming an electrode on an insulating substrate,
 applying the first liquid containing an enzyme to the electrode directly
 or via another layer and then drying it to form an immobilized enzyme
 layer, and applying the second liquid containing a polymer in which a
 pendant group having at least a fluoroalkylene block is attached to a
 non-fluorinated vinyl polymer, to the immobilized enzyme layer directly or
 via another layer and then drying it to form a permeation restricting
 layer. Preferably embodiments of "a polymer in which a pendant group
 having at least a fluoroalkylene block is attached to a non-fluorinated
 vinyl polymer" or others are as described for the first to fourth enzyme
 electrodes according to this invention; for example, polycarboxylic acid
 fluoroalcohol esters such as 1H,1H-perfluorooctyl polymethacrylate,
 1H,1H,2H,2H-perfluorodecyl polyacrylate, a copolymer of
 1H,1H-perfluorooctyl methacrylate and cyclohexyl methacrylate, and a
 copolymer of 1H,1H,2H,2H-perfluorodecyl acrylate and cyclohexyl
 methacrylate.
 The fifth enzyme electrode according to this invention comprises an
 electrode on an insulating substrate, an electrode protection layer mainly
 consisting of an urea compound, covering at least a part of the electrode,
 and an immobilized enzyme layer covering the electrode and the electrode
 protection layer.
 In this enzyme electrode, the electrode protection layer comprises an urea
 compound. Its content is not particularly limited, but the layer may
 substantially consist of the urea compound. The term, "mainly consist of"
 herein means that the content of the urea compound in the electrode
 protection layer is 50 wt % or higher.
 The electrode protection layer is preferably formed, covering the overall
 surface, but may cover a part of the electrode surface. There are no
 limitations for the thickness of the electrode protection layer. However,
 since an electrode protection layer consisting of urea formed by
 electrolysis may be sufficiently effective as described later in Examples,
 an average thickness corresponding to a ply of several molecules, for
 example, about 0.1 to 50 nm, may be adequate.
 The electrode protection layer and the immobilized enzyme layer in the
 enzyme electrode of this invention may be formed to be adjacent to each
 other directly or via an intervening layer. For example, a binding layer
 mainly consisting of a silane coupling agent is disposed between the
 electrode protection layer and the immobilized enzyme layer or an
 ion-exchange polymer layer mainly consisting of an ion-exchange polymer
 having a perfluorocarbon skeleton is disposed between the electrode
 protection layer and the immobilized enzyme layer. In this invention, an
 electrode on an insulating substrate is an electrode functioning at least
 as a working electrode.
 When a biosensor equipped with the fifth enzyme electrode is used, an urea
 compound may be added to a preserving solution or a calibration solution,
 to further improve stability in measurement.
 FIG. 10 shows an example of a biosensor employing an enzyme electrode of
 this invention, where the enzyme electrode is used as a working electrode
 7 and a counter electrode 8 and a reference electrode 9 are formed on a
 quartz substrate. The working electrode 7 and the counter electrode 8 are
 platinum electrodes, and the reference electrode 9 is a silver/silver
 chloride electrode. On the working electrode are sequentially formed a
 binding layer 3 mainly consisting of .gamma.-aminopropyltriethoxysilane,
 an immobilized enzyme layer 4 in which a catalytic enzyme is immobilized
 in an organic polymer molecule, and a permeation restricting layer 5
 consisting of a fluoroalcohol methacrylate resin. The working electrode 7,
 the counter electrode 8 and the reference electrode 9 are connected to
 corresponding measuring systems, respectively.
 This figure shows an example of an amperometric type of sensor, but the
 enzyme electrode of this invention may be, of course, applied to an
 ion-sensitive field effect transistor type of sensor.
 A biosensor according to this invention is particularly effective when used
 as an urinary-sugar sensor determining a glucose (urinary sugar) level in
 urine.
 A lower limit to urinary sugar is 50 mg/dL for a conventional sensor while
 the sensor of this invention can detect 1 to 5 mg/dL. In a conventional
 measuring apparatus, an S/N ratio is high so that when attempting to
 determine a low level of glucose below 50 mg/dL, a sensor output may be
 hidden by noises, making quantitative determination difficult. On the
 other hand, in a sensor according to this invention, an S/N ratio is
 adequately low to perform precise determination even in a low
 concentration range. Since a healthy individual has an urinary sugar value
 of 2 to 10 mg/dL, such improvement in measuring sensitivity is quite
 significant. Using a biosensor according to this invention, an urinary
 sugar level below 50 mg/dL can be quantitatively determined. An urinary
 sugar level can be, therefore, determined for an individual whose urinary
 sugar level is within a normal range or a prediabetic individual to
 collect data useful in prophylaxis for diabetes.
 When using a sensor according to this invention, influence of urea, vitamin
 C and acetaminophen contained in a large amount in urine may be
 effectively eliminated. Therefore, precise determination may be performed
 even after a subject takes a soft drink containing a large amount of
 vitamin C or an antipyretic containing acetaminophen.
 In a measuring apparatus according to this invention, a biosensor is
 preferably removable because it is desirable that the electrode of the
 biosensor is readily exchangeable since the electrode is consumable. Only
 the biosensor may be removable or wirings connecting the biosensor to
 other parts or a part comprising the biosensor may be removable. For
 example, in the measuring apparatus shown in FIG. 31, a wiring 54 between
 a biosensor 50 and an electrochemical measuring circuit 51 may be
 removable, or a part consisting of the biosensor 50, the wiring 54 and the
 electrochemical measuring circuit 51 may be removable.
 A data processor in a measuring apparatus according to this invention
 calculates a determination value based on an electric signal from a
 biosensor; for example, it operates by converting the electric signal to
 an analogue signal and/or a digital signal for calculating a determination
 value. The data processor may be equipped with a variety of means; for
 example, some or all of the following means;
 (a) a timer,
 (b) a time setting means for setting a time and a time indicator indicating
 a time at the time set by the time setting means,
 (c) an operation guide means describing operation instructions for the
 measuring apparatus,
 (d) a measured-value storing means for storing a calculated measured value,
 (e) a password registration means for registering a password for a user of
 the measuring apparatus,
 (f) a memo registration means for registering a memo,
 (g) an operation indicator for detecting malfunction in the measuring
 apparatus,
 (h) a calibration-timing indicator for detecting and indicating a
 calibration timing for the enzyme electrode,
 (i) an electrode-replacement-timing indicator for detecting and indicating
 a replacement timing for the enzyme electrode,
 (j) an abnormal-current indicator for detecting and indicating an abnormal
 current, and
 (k) an electrode calibrator for calibrating the enzyme electrode.
 Such a configuration may further improve operability. Processed results
 from one or more of (a) to (k) are indicated by the indicator.
 Embodiments of this invention will be further described with reference to
 the drawings.
 Embodiment 1
 The first embodiment will be described with reference to FIG. 1. The enzyme
 electrode of this embodiment comprises an electrode 2 as a working
 electrode on an insulating substrate 1, on which a binding layer 3 mainly
 consisting of .gamma.-aminopropyltriethoxysilane is formed. On the binding
 layer are sequentially formed an immobilized enzyme layer 4 in which a
 catalytic enzyme is immobilized 4a in an organic polymer and a permeation
 restricting layer 5 consisting of a fluoroalcohol methacrylate resin.
 The insulating substrate 1 may mainly consisting of a highly-insulative
 material such as ceramics, glass, quartz and plastics, which is preferably
 excellent in waterproof, heat resistance, chemical resistance and
 adhesiveness to an electrode.
 The electrode 2 may mainly consist of a material such as platinum, gold,
 silver and carbon; platinum is particularly preferable because of its
 excellent chemical resistance and detection property for hydrogen
 peroxide. The electrode 2 on the insulating substrate 1 may be formed by,
 for example, spattering, ion plating, vacuum deposition, chemical paper
 deposition and electrolysis; spattering is particularly preferable because
 the electrode 2 is highly adhesive to the insulating substrate 1 and a
 platinum layer can be easily formed. Furthermore, a titanium or chromium
 layer may be inserted between these layers for improving adhesiveness of
 the insulating substrate 1 to the electrode 2.
 The binding layer 3 on the electrode 2 may improve adhesiveness (binding
 strength) of the immobilized enzyme layer 4 to the insulating substrate 1
 and the electrode 2. It is also effective in improving wettability of the
 surface of the insulating substrate 1 and thickness uniformity during
 forming the immobilized enzyme layer 4 in which an enzyme is immobilized.
 It also exhibits selective permeation to ascorbic acid, uric acid and
 acetaminophen which may interfere with a reaction of hydrogen peroxide on
 the electrode 2. The binding layer 3 mainly consists of a silane coupling
 agent. Silane coupling agents which may be used include aminosilanes,
 vinylsilanes and epoxysilanes. .gamma.-aminopropyltriethoxysilane, an
 aminosilane, is particularly preferable in the light of adhesiveness and
 selective permeation. The binding layer 3 may be formed by, for example,
 spin coating of a silane coupling agent solution, where the concentration
 of the silane coupling agent is preferably about 1 v/v% (volume %) for
 significantly improving selective permeability.
 The immobilized enzyme layer 4 comprises an organic polymer base material
 in which a catalytic enzyme is immobilized. The immobilized enzyme layer 4
 may be formed by, for example, adding dropwise and applying by spin
 coating a solution containing some kind of enzyme, a protein cross-linking
 agent such as glutaraldehyde and albumin on the binding layer 3. Albumin
 may protect the enzyme from a reaction with the cross-linking agent and
 may be a protein base material. Enzymes to be immobilized include lactate
 oxidase, glucose oxidase, urate oxidase, galactose oxidase, lactose
 oxidase, sucrose oxidase, ethanol oxidase, methanol oxidase, starch
 oxidase, amino acid oxidase, monoamine oxidase, cholesterol oxidase,
 choline oxidase and pyruvate oxidase, which generate hydrogen peroxide as
 a product of their catalytic reaction or consume oxygen.
 Two or more enzymes may be used in combination for generating hydrogen
 peroxide; for example any combination of creatininase, creatinase and
 sarcosine oxidase for allowing creatinine to be detected.
 An enzyme may be combined with a coenzyme; for example, a combination of
 3-hydroxylactate dehydrogenase and nicotinamide adenine nucleotide (NAD)
 for allowing 3-hydroxylactic acid to be detected.
 An enzyme may be combined with an electron mediator, where an electron
 mediator which has been reduced by the enzyme is oxidized on the electrode
 surface to generate a current which is then measured. Such a combination
 may allow glucose to be detected.
 As described above, there are no limitations to the structure of the
 immobilized enzyme layer 4 as long as it contains at least an enzyme and
 can convert a target substance into an electrode sensitive substance such
 as hydrogen peroxide.
 There are no limitations to a process for forming the immobilized enzyme
 layer 4 as long as a uniform layer can be formed; screen printing may be,
 in addition to spin coating, used.
 A fluoroalcohol methacrylate resin composing of the permeation restricting
 layer 5 is a methacrylate resin whose carboxyl groups are partially or
 totally esterified by a fluoroalcohol. The fluoroalcohol is an alcohol,
 one or more or all of whose hydrogens are replaced with fluorine atoms.
 For example, 1H,1H-perfluorooctyl polymethacrylate or
 1H,1H,2H,2H-perfluorodecyl polyacrylate may be used. In this invention,
 for example, 1H,1H-perfluorooctyl polymethacrylate is a polymer in which
 methacrylic acid moieties are partially or totally esterified by
 1H,1H-perfluorooctylalcohol.
 The permeation restricting layer 5 may be formed by adding dropwise and
 applying by spin coating a solution of a fluoroalcohol methacrylate resin
 in a perfluorocarbon solvent such as perfluorohexane on the immobilized
 enzyme layer 4 in which a catalytic enzyme is immobilized.
 The concentration of the fluoroalcohol methacrylate resin in the solution
 may be preferably 0.1 to 5 wt %, more preferably about 0.3 wt %, depending
 on a target substance because a concentration within the range may, as
 described later, provide good permeation-restricting property (FIG. 6).
 There are no limitations to a process for forming the permeation
 restricting layer 5 as long as a uniform layer may be formed; spray
 coating or dipping may be, in addition to spin coating, employed.
 When the enzyme electrode of this embodiment is used as a glucose sensor,
 the outermost permeation restricting layer 5 restricts a diffusion rate of
 glucose and an organic polymer film 4 containing glucose oxidase
 catalytically reacts diffused glucose with oxygen to generate hydrogen
 peroxide and gluconolactone. A current generated when the hydrogen
 peroxide reaches the electrode 2 may be measured to determine a level of
 glucose. For an electrode system during measurement, a conventional
 external reference electrode is used in a two-electrode system, while both
 a counter electrode and a reference electrode are soaked in a measured
 solution at the same time for a three-electrode system.
 Embodiment 2
 This embodiment will be described with reference to FIG. 2. The enzyme
 electrode of this embodiment comprises an electrode 2 as a working
 electrode on an insulating substrate 1, on which a binding layer 3 mainly
 consisting of .gamma.-aminopropyltriethoxysilane is formed. On the binding
 layer are sequentially formed an ion-exchange polymer layer 6 mainly
 consisting of a perfluorocarbon-sulfonate acid polymer(NAFION), an
 immobilized enzyme layer 4 in which a catalytic enzyme is immobilized in
 an organic polymer, and a permeation restricting layer 5 consisting of a
 fluoroalcohol methacrylate resin.
 The electrode 2 and the y-aminopropyltriethoxysilane film 3 on the
 insulating substrate 1 are sequentially formed as described in Embodiment
 1.
 The ion-exchange polymer layer 6 mainly consisting of a
 perfluorocarbon-sulfonate acid polymer(NAFION) may be formed by, for
 example, adding dropwise and applying by spin coating a solution of a
 perfluorocarbon-sulfonate acid polymer in pure water and ethanol (50:50)
 on the binding layer 3 consisting of .gamma.-aminopropyltriethoxysilane.
 The solvent may be an alcohol such as isopropyl alcohol and ethanol. The
 concentration of the perfluorocarbon-sulfonate polymer is preferably 1 to
 10 w/v%, more preferably 5 to 7 w/v% because a concentration within the
 range may significantly contribute to eliminating influence of ascorbic
 acid interfering with an electrode reaction of hydrogen peroxide.
 Embodiment 3
 This embodiment will be described with reference to the drawings. As shown
 in FIG. 3, the enzyme electrode of this embodiment comprises a working
 electrode 7, a counter electrode 8 and a reference electrode 9 on an
 insulating substrate 1, on which a binding layer 3 mainly consisting of
 .gamma.-aminopropyltriethoxysilane is formed. On the binding layer are
 sequentially formed an immobilized enzyme layer 4 in which a catalytic
 enzyme is immobilized in an organic polymer, and a permeation restricting
 layer 5 consisting of a fluoroalcohol methacrylate resin. The materials
 for the working electrode 7 and the counter electrode 8 may be as
 described for the electrode 2 in Embodiments 1 and 2. The reference
 electrode 9 may be made of silver/silver chloride.
 Such a structure may allow a solution to be replaced while operating a
 sensor because the working electrode, the counter electrode and the
 reference electrode are formed on the single insulating substrate since
 the working electrode, the counter electrode and the reference electrode
 are electrically connected with each other as long as the surface of the
 sensor is wet and thus measurement can be continued even when the sensor
 is temporarily in contact with the air. It also allows precise
 electrochemical measurement by a three-electrode system. In particular, it
 may provide a small amperometric detection type of enzyme electrode.
 Furthermore, an ion-exchange polymer layer 6 consisting of a
 perfluorocarbon-sulfonate acid polymer may be formed between the
 .gamma.-aminopropyltriethoxysilane film 3 and the immobilized enzyme layer
 4 as described in Embodiment 2.
 Embodiment 4
 This embodiment will be described with reference to the drawings. As shown
 in FIG. 4, the enzyme electrode of this embodiment comprises two working
 electrodes 7, a counter electrode 8 and a reference electrode 9 on an
 insulating electrode 1, on which a binder layer 3 mainly consisting of
 .gamma.-aminopropyltriethoxysilane is formed. On these working electrodes
 7 are formed immobilized enzyme layers 4, 10 in which a different type of
 enzyme is immobilized, on which further a permeation restricting layer 5
 consisting of a fluoroalcohol methacrylate resin is formed. The materials
 for the working electrodes 7 and the counter electrode 8 may be as
 described for the electrode 2 in Embodiments 1 and 2. The reference
 electrode 9 is preferably made of silver/silver chloride.
 Such a configuration comprises layers in which a differently catalytic
 enzyme is formed, on two working electrodes, allowing a plurality of
 particular ingredients in a sample at the same time.
 Embodiment 5
 This embodiment is an example of a process for manufacturing an enzyme
 electrode according to this invention.
 First, a working electrode of platinum and a counter electrode and a
 reference electrode of silver/silver chloride are formed on a substrate of
 quartz.
 Then, the surfaces of each electrode and of the substrate are washed. The
 process may be conducted with, for example, an organic solvent or acid, an
 ultrasonic cleaner or combination thereof. A solvent or acid used should
 not cause damage to the electrode materials. The organic solvent is
 preferably a polar solvent; for example, ketones such as acetone and
 alcohols such as isopropyl alcohol. The acid is, for example, diluted
 sulfuric acid. Electrolysis cathode water, which is a solution generated
 around a cathode during electrolysis of pure water, may be used.
 Electrolysis cathode water is neutral to weakly alkaline and highly
 reductive, so that it can minimize damage to the substrate and the
 electrodes while making the potentials on the surfaces of the substrate
 and of adhered particles negative, to prevent detached particles from
 re-adhesion. Among the above procedures, a process of sequentially washing
 with acetone and sulfuric acid is preferable.
 Then, a binding layer is formed on the working electrodes, the counter
 electrode and the reference electrode. As described above, the binding
 layer may be preferably made of a silane coupling agent such as
 .gamma.-aminopropyltriethoxysilane.
 A coupling agent may be applied by, for example, spin coating, spraying,
 dipping and a hot stream method. Spin coating is a process that a solution
 or suspension of components for a binding layer such as a coupling agent
 is applied with a spin coater, by which a thinner binding layer may be
 formed, adequately controlling its thickness. Spraying is a process that
 for example, a solution of a coupling agent is sprayed on a substrate and
 dipping is a process that a substrate is soaked into, for example, a
 solution of a coupling agent. By these process, a binding layer may be
 formed by simple steps without no special apparatuses. A hot stream method
 is a process that for example, a stream of a coupling agent solution is
 flown over a substrate at an elevated temperature, by which a thinner
 binding layer may be formed, adequately controlling its thickness.
 After applying the coupling agent solution, the substrate is dried
 generally, but not limited to, at an ambient temperature (25.degree. C.)
 to 170.degree. C., for 0.5 to 24 hours depending on the temperature.
 Drying may be conducted in the air or in an inert gas such as nitrogen;
 for example, a nitrogen blowing may be employed, in which nitrogen is
 blown on the substrate to be dried.
 An enzyme solution is applied on the binding layer thus formed, to form an
 immobilized enzyme layer. The enzyme layer may be applied by, for example,
 spin coating or dipping (soaking), particularly spin coating because of
 its thickness controllability. After applying the enzyme solution, the
 substrate is dried at a temperature which does not impair enzyme activity,
 preferably an ambient temperature (25.degree. C.) to 40.degree. C., for
 0.5 to 24 hours depending on the drying temperature. Drying may be
 conducted in the air or in an inert gas such as nitrogen; for example, a
 nitrogen blowing may be employed, in which nitrogen is blown on the
 substrate to be dried.
 Then, on the substrate is applied, for example, a solution of a
 polycarboxylic acid fluoroalcohol ester to form a permeation restricting
 layer, by, for example, spin coating, dipping, spraying or brush coating,
 preferably spin coating because of its thickness controllability. By spin
 coating, a permeation restricting layer as a film about 0.01 to 3 .mu.m of
 thickness may be formed, adequately controlling its thickness.
 Alternatively, the substrate is soaked into the above solution by dipping
 for application thereof, and is then dried by nitrogen blowing, by which a
 permeation restricting layer may be formed by a simple process.
 After applying the solution, the substrate is dried at a temperature which
 does not impair enzyme activity, preferably an ambient temperature
 (25.degree. C.) to 40.degree. C., for 0.5 to 24 hours depending on the
 drying temperature. Drying may be conducted in the air or in an inert gas
 such as nitrogen; for example, a nitrogen blowing may be employed, in
 which nitrogen is blown on the substrate to be dried.
 As described above, an enzyme electrode may be manufactured, in which a
 variety of layers with particular functions are formed on an electrode.
 This embodiment shows a process that a binding layer, an immobilized
 enzyme layer and a permeation restricting layer are formed on all of
 working electrodes, a counter electrode and a reference electrode, but
 this invention is not limited to the configuration. It is, for example,
 possible to form a binding layer, an immobilized enzyme layer and a
 permeation restricting layer on working electrodes and a counter electrode
 while forming a binder layer on a reference layer and then a protection
 layer for protecting the reference layer. Although this embodiment shows a
 three-electrode system of biosensor comprising working electrodes, a
 counter electrode and a reference electrode, a configuration that a
 working electrode of platinum and a reference electrode are formed on a
 quartz substrate may be acceptable.
 Embodiment 6
 This embodiment will be described with reference to the drawings. The
 enzyme electrode of this embodiment, as shown in FIG. 16, comprises an
 electrode 2 as a working electrode on an insulating substrate 1, and an
 electrode protection layer 18 mainly consisting of an urea compound which
 covers the surface of the electrode. The electrode protection layer 18 is
 selectively formed on the surface of the electrode 2. On the overall
 surface are formed a binding layer 3 mainly consisting of
 .gamma.-aminopropyltriethoxysilane and then an immobilized enzyme layer 4
 in which an enzyme is immobilized in an organic polymer as a base
 material.
 The insulating substrate 1 may mainly consisting of a highly-insulative
 material such as ceramics, glass, quartz and plastics, which is preferably
 excellent in waterproof, heat resistance, chemical resistance and
 adhesiveness to an electrode.
 The electrode 2 may mainly consist of a material such as platinum, gold,
 silver and carbon; platinum is particularly preferable because of its
 excellent chemical resistance and detection property for hydrogen
 peroxide. The electrode 2 on the insulating substrate 1 may be formed by,
 for example, spattering, ion plating, vacuum deposition, chemical paper
 deposition and electrolysis; spattering is particularly preferable because
 the electrode 2 is highly adhesive to the insulating substrate 1 and a
 platinum layer can be easily formed. Furthermore, a titanium or chromium
 layer may be inserted between these layers for improving adhesiveness of
 the insulating substrate 1 to the electrode 2.
 The electrode protection layer 18 covering the electrode 2 restricts
 permeation of contaminants such as urea contained in a sample to the
 electrode. The electrode protection layer 18 comprises an urea compound
 such as, but not limited to, urea and thiourea, preferably urea because of
 its lower toxicity and lower cost. The enzyme electrode of this embodiment
 comprises the electrode protection layer containing a contaminant on the
 surface of the electrode for preventing variation of sensitivity due to
 contamination during operation. It will be, therefore, readily understood
 in the light of the function of the electrode protection layer that the
 urea compound is not limited to the above specific examples.
 The electrode protection layer 18 may be formed by, for example, dipping,
 plasma polymerization and electrolysis, preferably electrolysis which may
 be conducted with an inexpensive apparatus for a reduced process time.
 Specifically, it is preferable to form an electrode protection layer by
 soaking an insulating substrate on which an electrode has been formed into
 a mixed solution containing a supporting electrolyte and an urea compound
 and applying electricity to the solution, where the concentration of urea
 in the mixed solution is preferably 0.1 mM to 6.7 M, more preferably 1 M
 to 6.7 M. The concentration of sodium chloride in the mixed solution is
 preferably 0.1 mM to 2 M, more preferably 1.5 mM to 150 mM. Thus, there
 may be provided a high-quality electrode protection layer which can
 effectively restrict adhesion of contaminants to the electrode and
 restrict permeation of interferent substances for good selectivity. It may
 improve adhesiveness to the binding layer 3 mainly consisting of
 .gamma.-aminopropyltriethoxysilane.
 The binding layer 3 on the electrode protection layer 18 may improve
 adhesiveness (binding strength) of the immobilized enzyme layer 4 thereon
 to the insulating substrate 1 and the electrode protection layer 18. It is
 also effective in improving wettability of the surface of the insulating
 substrate 1 and thickness uniformity during forming the immobilized enzyme
 layer 4 in which an enzyme is immobilized. It also exhibits selective
 permeation to ascorbic acid, uric acid and acetaminophen which may
 interfere with a reaction of hydrogen peroxide on the electrode 2. The
 binding layer 3 mainly consists of a silane coupling agent. Silane
 coupling agents which may be used include aminosilanes, vinylsilanes and
 epoxysilanes. y-aminopropyltriethoxysilane, an aminosilane, is
 particularly preferable in the light of adhesiveness and selective
 permeation. The binding layer 3 may be formed by, for example, spin
 coating of a silane coupling agent solution, where the concentration of
 the silane coupling agent is preferably about 1 v/v% (volume %) for
 significantly improving selective permeability.
 The immobilized enzyme layer 4 comprises an organic polymer base material
 in which a catalytic enzyme is immobilized. The immobilized enzyme layer 4
 may be formed by, for example, adding dropwise and applying by spin
 coating a solution containing some kind of enzyme, a protein cross-linking
 agent such as glutaraldehyde and albumin on the binding layer 3. Albumin
 may protect the enzyme from a reaction with the cross-linking agent and
 may be a protein base material. Enzymes to be immobilized include lactate
 oxidase, glucose oxidase, urate oxidase, galactose oxidase, lactose
 oxidase, sucrose oxidase, ethanol oxidase, methanol oxidase, starch
 oxidase, amino acid oxidase, monoamine oxidase, cholesterol oxidase,
 choline oxidase and pyruvate oxidase, which generate hydrogen peroxide as
 a product of their catalytic reaction or consume oxygen.
 Two or more enzymes may be used in combination for generating hydrogen
 peroxide; for example any combination of creatininase, creatinase and
 sarcosine oxidase for allowing creatinine to be detected.
 An enzyme may be combined with a coenzyme; for example, a combination of
 3-hydroxylactate dehydrogenase and nicotinamide adenine nucleotide (NAD)
 for allowing 3-hydroxylactic acid to be detected.
 An enzyme may be combined with an electron mediator, where an electron
 mediator which has been reduced by the enzyme is oxidized on the electrode
 surface to generate a current which is then measured. Such a combination
 may allow glucose to be detected.
 As described above, there are no limitations to the structure of the
 immobilized enzyme layer 4 as long as it contains at least an enzyme and
 can convert a target substance into an electrode sensitive substance such
 as hydrogen peroxide.
 There are no limitations to a process for forming the immobilized enzyme
 layer 4 as long as a uniform layer can be formed; screen printing may be,
 in addition to spin coating, used.
 Embodiment 7
 This embodiment will be described with reference to the drawings. The
 enzyme electrode of this embodiment, as shown in FIG. 17, comprises an
 electrode 2 as a working electrode on an insulating substrate 1, and an
 electrode protection layer 18 mainly consisting of an urea compound which
 covers the surface of the electrode. On the overall surface are
 sequentially formed a binding layer 3 mainly consisting of
 y-aminopropyltriethoxysilane, an ion-exchange polymer layer 6 consisting
 of a perfluorocarbon-sulfonate acid polymer and an immobilized enzyme
 layer 4 in which an enzyme is immobilized in an organic polymer as a base
 material.
 The electrode 2, the electrode protection layer 18, the
 y-aminopropyltriethoxysilane layer 3 and the immobilized enzyme layer 4
 are sequentially formed on the insulating substrate 1 as described in
 Embodiment 1.
 The perfluorocarbon-sulfonate polymer composing of the ion-exchange polymer
 layer 6 may be, for example, NAFION, which is a cation-exchange polymer in
 which perfluoroalkylene ether side chains having a terminal sulfonic group
 are attached to a perfluoromethylene principal chain (Formula 1).
 ##STR4##
 The ion-exchange layer 6 such as a NAFION film disposed on the immobilized
 enzyme layer may eliminate influence of interferent substances, which may
 be synergic with the permeation-restricting effect of the electrode
 protection layer 3 to interferent substances to significantly minimize the
 influence of interferent substances.
 The ion-exchange polymer layer 6 may be formed by adding dropwise and
 applying by spin coating a solution of NAFION in pure water and ethanol
 (50:50) on the .gamma.-aminopropyltriethoxysilane layer 3. The solvent may
 be an alcohol such as isopropyl alcohol and ethanol. The concentration of
 NAFION is preferably 1 to 10 w/v%, more preferably 5 to 7 w/v% because a
 concentration within the range may significantly contribute to eliminating
 the influence of interferent substances.
 Embodiment 8
 This embodiment will be described with reference to the drawings. The
 enzyme electrode of this embodiment, as shown in FIG. 18, comprises an
 electrode 2 as a working electrode on an insulating substrate 1, and an
 electrode protection layer 18 mainly consisting of an urea compound which
 covers the surface of the electrode. On the overall surface are
 sequentially formed a binding layer 3 mainly consisting of
 .gamma.-aminopropyltriethoxysilane, an ion-exchange polymer layer 6
 consisting of a perfluorocarbon-sulfonate acid polymer, an immobilized
 enzyme layer 4 in which an enzyme is immobilized in an organic polymer as
 a base material and a permeation restricting layer 5 as the uppermost
 layer. The electrode 2, the electrode protection layer 18, the
 .gamma.-aminopropyltriethoxysilane layer 3, the ion-exchange polymer layer
 6 consisting of a perfluorocarbon-sulfonate polymer and the immobilized
 enzyme layer 4 in which an enzyme is immobilized are sequentially formed
 on the insulating substrate 1 as described in Embodiments 1 and 2.
 The permeation restricting layer 5 may be made of a polymer in which a
 pendant group containing at least a fluoroalkylene block is attached to a
 non-fluorinated vinyl polymer. The permeation restricting layer 5 may be
 formed by applying by spin coating the above polymer solution. For
 example, a solution of a fluoroalcohol polymethacrylate in a
 perfluorocarbon solvent such as perfluorohexane may be added dropwise and
 applied by spin coating on the immobilized enzyme layer 4 in which a
 catalytic enzyme is immobilized. The ester concentration in the solution
 may be preferably 0.1 to 5 wt %, more preferably about 0.3 wt %, depending
 on a target substance because a concentration within the range may, as
 described later, provide good permeation-restricting property.
 There are no limitations to a process for forming the permeation
 restricting layer 5 as long as a uniform layer may be formed; spray
 coating or dipping may be, in addition to spin coating, employed.
 The permeation restricting layer 5 consisting of a polymer having the
 particular structure may prevent adhesion of contaminants such as proteins
 and urea compounds, which is synergic with prevention of contaminant
 adhesion by the electrode protection layer 3, leading to stable output
 properties even for a long-term use. Furthermore, it may provide good
 permeation restricting- property, resulting in significant extension of
 measurable concentration range. Furthermore, good adhesiveness between the
 permeation-restriction layer 5 and the immobilized enzyme layer 4 may
 permit to consistently measure a target substance in a solution for a long
 time and to consistently measure an ionized substance such as lactic acid
 because the permeation restricting layer has no charges and therefore
 little interacts with an ionic substance.
 When the sensor of this embodiment is used as a glucose sensor, the
 outermost permeation restricting layer 5 restricts a diffusion rate of
 glucose and an immobilized enzyme layer 4 containing glucose oxidase
 catalytically reacts diffused glucose with oxygen to generate hydrogen
 peroxide and gluconolactone. A current generated when the hydrogen
 peroxide reaches the electrode 2 may be measured to determine a level of
 glucose. For an electrode system during measurement, a conventional
 external reference electrode is used in a two-electrode system, while both
 a counter electrode and a reference electrode are soaked in a measured
 solution at the same time for a three-electrode system.
 Embodiment 9
 This embodiment will be described with reference to the drawings. The
 enzyme electrode of this embodiment, as shown in FIG. 19, comprises a
 working electrode 7, a counter electrode 8 and a reference electrode 9. On
 the working electrode 7 and the counter electrode 8 are sequentially
 formed an electrode protection layer 18, a binding layer 3 mainly
 consisting of .gamma.-aminopropyltriethoxysilane, an ion-exchange polymer
 layer 6 mainly consisting of a perfluorocarbon-sulfonate polymer, an
 immobilized enzyme layer 4 in which a catalytic enzyme is immobilized and
 a permeation restricting layer 7. The materials for the working electrode
 7 and the counter electrode 8 may be the same as that for the electrode 2.
 The reference electrode 9 may be made of silver/silver chloride.
 Such a structure may allow a solution to be replaced while operating a
 sensor because the working electrode, the counter electrode and the
 reference electrode are formed on the single insulating substrate since
 the working electrode, the counter electrode and the reference electrode
 are electrically connected with each other as long as the surface of the
 sensor is wet and thus measurement can be continued even when the sensor
 is temporarily in contact with the air. It also allows precise
 electrochemical measurement by a three-electrode system. In particular, it
 may provide a small amperometric detection type of enzyme electrode.
 Embodiment 10
 FIG. 28 shows a sensor employing an enzyme electrode according to this
 invention. In the embodiment, a working electrode 7 is an enzyme electrode
 and a counter electrode 8 and a reference electrode 9 are formed on a
 quartz substrate. The working electrode 7 and the counter electrode 8 are
 made of platinum while the reference electrode 9 is made of silver/silver
 chloride. The working electrode 7 is completely covered by an electrode
 protection layer 18 consisting of urea, on which are sequentially formed a
 binding layer 3 mainly consisting of .gamma.-aminopropyltriethoxysilane,
 an ion-exchange polymer layer 6 consisting of NAFION, an immobilized
 enzyme layer 4 and a permeation restricting layer 5 consisting of a
 fluoroalcohol methacrylate resin. The working electrode 7, the counter
 electrode 8 and the reference electrode 9 are connected to corresponding
 measuring systems, respectively.
 In this embodiment, the working electrode 7, the counter electrode 8 and
 the reference electrode 9 are formed on the single insulating substrate 1,
 but these electrodes may be formed on multiple substrates. FIG. 29(a)
 shows a configuration where a working electrode 7, a counter electrode 8
 and a reference electrode 9 are separately formed on different insulating
 substrates 1, while FIG. 29(b) shows a configuration where a working
 electrode 7 and a counter electrode 8 are formed on the same insulating
 substrate 1 and a reference electrode 9 is on another insulating substrate
 1.
 These figures show examples of an amperometric type of sensor, but the
 enzyme electrode of this invention may be, of course, applied to an
 ion-sensitive field effect transistor type of sensor.
 Embodiment 11
 This embodiment is an example of a measuring apparatus according to this
 invention equipped with a biosensor, an electrochemical measuring circuit,
 a data processor and a data indicator, which will be described with
 reference to FIGS. 31 and 32.
 The measuring apparatus, as shown in FIG. 31, comprises a biosensor 50, an
 electrochemical measuring circuit 51, a data processor 52 and a data
 indicator 53, which are connected by wirings 54.
 The biosensor 50 may comprises an enzyme electrode, for example, described
 in any of Embodiments 1 to 4. Since the biosensor 50 is consumable, it is
 preferably removable for facilitating replacement.
 The electrochemical measuring circuit 51 is a potentiostat in this
 embodiment, but it may be any circuit which may apply a constant potential
 to the biosensor 50 to determine a current value.
 The data processor 52 has a configuration shown in FIG. 32, comprising a
 timer 60, a time setting means 61, a time indicator 62, an operation guide
 means 63, a measured-value storing means 64, a password registration means
 65, a memo registration means 66, an operation indicator 67, a
 calibration-timing indicator 68, an electrode-replacement-timing indicator
 69, an abnormal-current indicator 70 and an electrode calibrator 71. The
 processor comprising these means may allow an operator to smoothly conduct
 calibration of the electrode, measurement and storage of measurement data.
 Although this embodiment has a personal computer (hereinafter, referred to
 as a "PC") as a data processor 52, it may be any apparatus having an
 operation unit such as a microprocessor which may process a signal from
 the electrochemical circuit 51. A signal processed by the data processor
 52 is converted into a measured value, which is then indicated by the data
 indicator 53.
 In this embodiment, the data indicator 53 is a display for PC, but it may
 be any apparatus which may indicate data processed by the data processor
 52. The data processed by the data processor 52 may include a measured
 value calculated by the data processor 52, status (normal or malfunction)
 of the biosensor 50, detection results for an abnormal current value, a
 timing for replacing the biosensor 50, a timing and a procedure for
 calibration of the biosensor 50, a date, a time, a clock, a signal from
 the electrochemical measuring circuit 51 which is processed by the
 operation unit in the data processor 52, instructions for operation means
 for initial setting, and instructions giving an advice during operation.
 An indicating means may be a digital number, an analogue number or voice.
 Other indicating means may include beep, light, vibration, color, graphic
 and heat, but a digital or analogue number is preferable.
 The wiring 54 may be any electric wire which can connect these.
 Next, each means in the data processor 52 (FIG. 32) will be described.
 The timer 60 is a clock built in a PC, but it may be any type giving a time
 to the operation unit.
 The time setting means 61 sets a time when a measurement is performed,
 using the timer 60. In this embodiment, the means utilizes some functions
 of the clock built in the PC as is for the timer 60, but it may be any
 type which can give a time to the operation unit as well as set a time of
 measurement. It is preferable that a plurality of times can be set for
 facilitating multiple measurements a day. It may be more convenient that
 an operator can select whether using the time setting means 61 or not.
 The time indicator 62 indicates a time set by the time setting means 61.
 For example, the time setting means 61 set to indicate a time every 12
 hours allows an operator to know a measuring time every 12 hours from the
 time indicator 62.
 The operation guide means 63 describes an operation procedure for a
 measuring apparatus or instructions for operation. Use of the operation
 guide means 63 can be selected by setting as appropriate.
 The measured-value storing means 64 stores a measured value from the
 measuring apparatus or other information. Its semiconductor storage
 element is preferably a RAM (random access memory). The measured-value
 storing means 64 can preferably store a plurality of measured values. The
 measured-value storing means 64 can store not only a measured value, but
 also a variety of information to be processed by the data processor 52.
 Data to be stored can be controlled by setting as appropriate.
 The password registration means 65 controls use of the measuring apparatus
 by an individual other than a particular operator and data on measured
 values for allowing user's privacy to be protected. A password is
 preferably constructed with a four or more digit number or alphanumeric
 for ensuring higher security. The password registration means 65 can
 preferably register a plurality of passwords for protecting two or more
 users' privacy. In this embodiment, any data cannot be input/output
 without a four digit password although use of the password registration
 means 65 can be appropriately selected by setting.
 Preferably, the memo registration means 66 comprises a memo register for
 registering a memo, a memo itemizing means for accessing a registered memo
 group, a memo selector for selecting a memo item to be registered from the
 accessed memo group and a memo access means for accessing the memo
 selected by the memo selector. In this embodiment, the memo registration
 means having the configuration can register subject's data such as weight,
 blood pressure and temperature at measurement. Use of the memo
 registration means 66 can be appropriately selected by setting.
 The operation indicator 67 indicates a status when one or both of the
 wirings 54 between the biosensor 50 and the electrochemical measuring
 circuit 51 and between the electrochemical measuring circuit 51 and the
 data processor 52 are disconnected. Use of the operation indicator 67 can
 be appropriately selected by setting.
 The calibration-timing indicator 68 indicates a timing for calibration of
 the biosensor 50. After being used to a certain extent, an enzyme
 electrode should be calibrated. Thus, the calibration timing indicator 68
 indicates a calibration timing. The timing may be determined, based on an
 accumulated measuring time or the number of measurement. In this
 embodiment, one or both of the criteria can be selected as criteria for
 calibration, by setting.
 The electrode-replacement-timing indicator 69 indicates timing for
 replacing an electrode in the biosensor 50. The timing may be determined,
 based on some criteria such as an accumulated measuring time, the number
 of measurement and voltage drop in a battery. In this embodiment, one or
 all of the factors can be selected as criteria for calibration, by
 setting.
 The abnormal-current indicator 70 indicates a status when measurement
 cannot be conducted due to an abnormal current passing through the
 biosensor 50, the electrochemical measuring circuit 51, the data processor
 52 and/or the wirings 54 connecting these or when some of these elements
 are broken.
 "Indication" in the operation indicator 67, the calibration-timing
 indicator 68, the electrode-replacement-timing indicator 69 and the
 abnormal current indicator 70 may be, for example, performed through the
 above data indicator to inform certain data of a measuring apparatus
 operator.
 The electrode calibrator 71 is used during an initial stage of use or
 calibration. It can indicate a calibration procedure for the biosensor 50
 and calibrate the biosensor 50. The calibration procedure may be indicated
 through the calibration-timing-indicator 68.
 The measuring apparatus of this embodiment indicates various data such as
 the lifetime of a biosensor, a timing of calibration and an operation
 procedure for the device, so that even an unfamiliar individual can
 conduct precise measurement.
 Although this embodiment has a configuration where the biosensor 50, the
 electrochemical measuring circuit 51, the data processor 52 and the data
 indicator 53 are connected via the wirings 54, an alternative
 configuration may be employed, in which an electrochemical measuring
 circuit 51 and a data indicator 53 are directly connected without a data
 processor 52. In such a configuration, an analogue signal from the
 biosensor 50 is directly sent to the data indicator 53, which then
 indicates a measured value via, for example, an indicating system of a
 graduation and a pointer. It may be helpful to provide a table for
 converting an indicated value into an urinary-sugar or blood-sugar value.
 Embodiment 12
 This embodiment relates to a measuring apparatus as shown in FIG. 31 which
 is further equipped with a temperature sensor 56 and a pH sensor 57. It
 will be described with reference to FIG. 33.
 As shown in FIG. 33, the measuring apparatus comprises a biosensor 50, an
 electrochemical measuring circuit 51, a data processor 52, a data
 indicator 53, a temperature sensor 56 and a pH sensor 54, which are
 connected via wirings 54.
 The data processor 52 processes an electric signal from the temperature
 sensor 56 and the pH sensor 57 to calculate the temperature and the pH.
 Then, a measured value for a particular component in a sample estimated in
 the data processor 52 is corrected on the basis of the temperature and the
 pH to indicate the corrected data by the data indicator 53.
 The temperature sensor 56 may be any type which can provide a form of data
 processable by the data processor 52, preferably a thermoelectric
 thermometer or resistance thermometer. The temperature sensor 56 measures
 a sample or ambient temperature. When measuring a sample temperature, the
 temperature sensor 56 is formed on the substrate comprising the biosensor
 using an enzyme electrode, for precisely determining the sample
 temperature and accurately correcting a measured value in detecting a
 particular component in the sample. When the measuring apparatus has the
 biosensor and the temperature sensor separately, these sensors may be
 soaked in the sample at the same time, for eliminating necessity for
 replacing the temperature sensor and the biosensor as one unit, leading to
 cost reduction. When measuring an ambient temperature, the temperature
 sensor 56 separately formed from the biosensor is placed in an ambient.
 The temperature sensor 56 is placed, for example, within the data
 indicator 53 or the electrochemical measuring circuit 51, for facilitating
 monitoring whether the ambient temperature is within measurable limits.
 FIG. 30 and FIG. 15 show examples of a measuring apparatus in which a
 temperature sensor 56 is formed on a substrate comprising a biosensor. The
 measuring apparatus shown in FIG. 30 comprises a working electrode 7, a
 counter electrode 8, a reference electrode 9 and also a temperature sensor
 56. On the working electrode 7, the counter electrode 8 and the
 temperature sensor 56 are sequentially formed a binding layer 3, an
 immobilized enzyme layer 4 and a permeation restricting layer 5. On the
 reference electrode 9 are formed a binder layer 3 and a protection layer
 20. Such a configuration may allow a measured value to be accurately
 corrected on the basis of a temperature.
 The pH sensor 57 is preferably, but not limited to, a glass electrode or
 ion-sensitive field effect transistor. The pH sensor 57 may be calibrated
 using a solution of a pH indicator in a calibration liquid for configuring
 the biosensor 50, for allowing calibration of the biosensor 50 and the pH
 sensor 57 at the same time. The pH indicator may be preferably an oxalate
 or phthalate solution which is used in a common glass pH meter.
 The measuring apparatus of this embodiment may permit to accurately
 determine a level of a particular component in a sample in a wide
 temperature or pH range because a measured value from the enzyme electrode
 can be corrected using a temperature and a pH for each sample.
 Although this embodiment has a configuration where the temperature sensor
 56 and the pH sensor 57 are connected to the data processor 52, these may
 be connected to an electrochemical measuring circuit 51.
 Embodiment 13
 This embodiment relates to a measuring apparatus as shown in FIG. 33,
 further comprising a communication processor connected to a data
 processor, which transmits data from the data processor to an external
 device. It will be described with reference to FIG. 34.
 The measuring apparatus of this embodiment, as shown in FIG. 34, comprises
 a data processor 52 and a communication processor 58 which are mutually
 connected via a wiring 54. The communication processor 58 transmits
 information on a measured value to an external device. It is commonly a
 modem, but any device capable of communication processing may be used. A
 communication circuit used for communication may be, but not limited to, a
 telephone line, an infrared system or a wireless telephone. Information to
 be transmitted include those processed in the data processor 52 and those
 indicated by the data indicator 53. The communication processor 58 can
 transmit, for example, a current value in the biosensor 50, a password, a
 pH, a temperature, notes, a measured value calculated in the data
 processor 52, a timing for replacing the biosensor 50, a timing for
 calibration of the biosensor 50, data for checking operation or
 malfunction of the biosensor 50, an abnormal current and a signal from the
 electrochemical measuring circuit 51 processed by the operation unit of
 the data processor 52, to an external device such as a server or computer
 connected to the apparatus via a communication circuit. Information to be
 transmitted may be selected by setting as appropriate.
 The measuring apparatus of this embodiment may be used to determine an
 urinary-sugar level in a patient with diabetes by him/herself at home,
 which may be then transmitted to a medical institute such as a hospital
 via a telephone line. It, therefore, may allow the patient to be
 appropriately advised in terms of diet or exercise control by the
 institute. Thus, it may allow administrating a patient with diabetes at
 home. Furthermore, since the apparatus can transmit data on malfunction of
 the enzyme electrode, services such as repair or maintenance of the
 apparatus from its manufacturer as appropriate.
 Embodiment 14
 This embodiment relates to a measuring apparatus as shown in FIG. 34
 further comprising a printer 59, which will be described with reference to
 FIG. 35.
 The measuring apparatus of this embodiment, as shown in FIG. 35, comprises
 a data processor 52 and a printer 59 which are mutually connected via a
 wiring 54. The printer 59 may be, but not limited to, a thermal, heat
 transfer, dot impact, inkjet or laser-beam dry printer, preferably a
 thermal printer which is of low cost and simple. The wiring 54 connecting
 the printer 59 to the data processor 52 may be an infrared ray, rather
 than an electric cord, taking an operation mode without a printer 59 into
 account. The printer 59 may be any printer which can print data to be
 indicated in the data indicator 53, but it may be set to print selected
 data.
 The measuring apparatus of this embodiment allows data such as a measured
 value to be printed on a paper for storage, and also makes it possible
 that a patient with diabetes can use its printing function to print
 determination results on a paper, which the patient then brings to a
 physician for obtaining appropriate advice from the physician.
 Embodiment 15
 This embodiment relates to a measuring apparatus as shown in FIG. 35
 further comprising an external storage 55, which will be described with
 reference to FIG. 36.
 The measuring apparatus of this embodiment, as shown in FIG. 36, comprises
 a data processor 52 and an external storage which are mutually connected
 via a wiring 54. The external storage 55 may be a common storage medium;
 preferably magnetic storage media such as a floppy disk, semiconductor
 storage media such as a memory card, and optical storage media such as an
 optical disk because they are readily removed and inexpensive.
 The measuring apparatus of this embodiment may be used to store measuring
 data on a storage medium, which a user can, as necessary, bring to a
 hospital. A physician in the hospital can analyze the stored measuring
 data to give an appropriate medical treatment to the patient with
 diabetes. Furthermore, a mass of measuring data can be stored for a long
 time. The apparatus is administered by means of passwords, so that
 patient's privacy can be protected and one apparatus may be used by two or
 more users. Data to be stored may be selected by setting as appropriate.
 EXAMPLES
 This invention will be specifically described with reference to Examples.
 Example 1
 On a 10 mm.times.6 mm quartz substrate were formed a working electrode of
 platinum (area: 7 mm.sup.2), a counter electrode (area: 4 mm.sup.2) and a
 reference electrode of silver/silver chloride (area: 1 mm.sup.2).
 Then, on the overall surface was applied by spin coating a 1 v/v% solution
 of .gamma.-aminopropyltriethoxysilane to form a binding layer, on which
 was applied by spin coating a 22.5 w/v% solution of albumin containing
 glutaraldehyde at 1 v/v%, to form an immobilized enzyme layer.
 Then, the following two types of enzyme electrodes were manufactured, with
 different structures of the outermost layer (permeation restricting
 layer).
 (1) On the overall surface of the immobilized enzyme layer was applied by
 spin coating and then dried a 0.3 wt % solution of a fluoroalcohol
 methacylate resin in perfluorohexane, to form a permeation restricting
 layer for providing the first enzyme electrode. Spin coating was conducted
 under the conditions of 3000 rpm and 30 sec. The fluoroalcohol
 methacrylate resin was Florard 722 (Sumitomo 3M), 1H,1H-perfluorooctyl
 polymethacrylate with an average molecular weight (Mn) of about 7000.
 Perfluorohexane as a diluent was Florard 726 (Sumitomo 3M). In addition,
 in a similar manner, on a quartz substrate was directly applied by spin
 coating a fluoroalcohol methacrylate resin to give a sample, whose
 thickness was then determined. After the film formation, a part of the
 film was removed with an ultrasonic cutter to provide a corrugated
 surface. Then, the corrugated surface was subject to observation with an
 atomic force microscope(AFM) to determine the film thickness. The
 thickness of the fluoroalcohol methacrylate was about 50 nm.
 (2) On the overall surface of the immobilized enzyme layer was applied by
 spin coating and dried a 10 w/v% solution of a polyalkylsiloxane in
 toluene to form a permeation restricting layer for providing the second
 enzyme electrode. The polyalkylsiloxane was Pergan Z (Dow Corning).
 Two sensors comprising one of the first and the second enzyme electrodes
 thus manufactured were soaked for storing in a buffer solution of TES
 (Methyl N-tris(hydroxymethyl)-2-aminoethanesulfonate), pH 7, containing
 sodium chloride at 150 mM, while a 200 mg/dL standard solution of glucose
 was measured with these sensors once a day for 20 days. FIG. 5 shows
 outputs of these sensors for the glucose solution as relative values where
 the initial outputs were regarded as 100%. Table 1 shows comparison data
 on frequencies of cracks by observing the surface of the permeation
 restricting layer with a scanning electron microscope after a given period
 of use.
 For the sensor comprising the polyalkylsiloxane, the sensor output has been
 increased from Day 7 (FIG. 5). Furthermore, in comparison with the initial
 state, there were observed cracks in the film, which were increased as the
 time elapsed (Table 1). In contrast, the sensor comprising the
 fluoroalcohol methacrylate resin gave stable outputs at least for 20 days,
 with no observed cracks.
 TABLE 1
 Cracks in a permeation restricting layer surface
 Initial Day 20
 Sensor comprising a fluoroalcohol methacrylate resin
 Without an enzyme film 1 1
 With an enzyme film 1 1
 Sensor comprising a polyalkylsiloxane
 Without an enzyme film 2 3
 With an enzyme film 2 5
 Crack frequencies were categorized into five levels according to the number
 of cracks observed in an area of 1 cm.times.1 cm.
 1: Not detectable
 2: Low (several cracks)
 3: Moderate (several tens of cracks or one large crack)
 4: High (several hundreds of cracks or several large cracks)
 5: Severe (several tens of large cracks)
 The results in Table 1 indicate that increase of a relative sensor output
 might be due to cracks in the permeation restricting layer. The cracks in
 the permeation restricting layer probably generated because the layer
 could not endure expansion of the lower immobilized enzyme layer. The
 results in this example shows that a permeation restricting layer
 consisting of a fluoroalcohol methacrylate resin is adequately strong to
 endure expansion of the lower enzyme film.
 Although this example has illustrated a sensor comprising a working
 electrode, a counter electrode and a reference electrode, on whose overall
 surface were formed a binding layer, an immobilized enzyme layer and a
 permeation restricting layer, these layers may be formed only on the
 working electrode.
 Example 2
 On a 10 mm.times.6 mm quartz substrate were formed a working electrode of
 platinum (area: 7 mm.sup.2), a counter electrode (area: 4 mm.sup.2) and a
 reference electrode of silver/silver chloride (area: 1 mm.sup.2).
 Then, on the overall surface was applied by spin coating a 1 v/v% solution
 of y-aminopropyltriethoxysilane to form a binding layer, on which was
 applied by spin coating a 22.5 w/v% solution of albumin containing
 glutaraldehyde at 1 v/v%, to form an immobilized enzyme layer.
 Then, on the immobilized enzyme layer was applied by spin coating and then
 dried a 0.3 wt % solution of a fluoroalcohol acrylate resin in
 hexafluoroxylene, to form a permeation restricting layer for providing the
 first enzyme electrode. Spin coating was conducted under the conditions of
 3000 rpm and 30 sec. The applied solution was prepared by diluting a
 solution of 1H,1H,2H,2H-perfluorodecyl polyacrylate (acrylate
 resin/hexafluoroxylene=17/83, viscosity: 20 cps at 25.degree. C.) with
 hexafluoroxylene to the indicated concentration.
 The sensor comprising the enzyme electrode thus manufactured was soaked for
 storing in a buffer solution of TES (Methyl
 N-tris(hydroxymethyl)-2-aminoethanesulfonate), pH 7, containing sodium
 chloride at 150 mM, while a 200 mg/dL standard solution of glucose was
 measured with the sensor once a day for 20 days. The surface of the
 permeation restricting layer was observed with a scanning electron
 microscope for crack generation. The results showed that the sensor of
 this embodiment gave stable outputs at least for 20 days, with no observed
 cracks in the permeation restricting layer, as was in the sensor of
 Example 1 comprising a fluoroalcohol acrylate resin.
 Example 3
 On a 10 mm.times.6 mm quartz substrate were formed a working electrode of
 platinum (area: 7 mm.sup.2), a counter electrode (area: 4 mm.sup.2) and a
 reference electrode of silver/silver chloride (area: 1 mm.sup.2).
 Then, on the overall surface was applied by spin coating a 1 v/v% solution
 of y-aminopropyltriethoxysilane to form a binding layer, on which was
 applied by spin coating a 5 w/v% solution of perfluorocarbon-sulfonate
 polymer, to form an ion-exchange polymer layer mainly consisting of the
 perfluorocarbon-sulfonate polymer (NAFION) and then applied by spin
 coating a 22.5 w/v% solution of albumin containing glutaraldehyde at 0.5
 v/v% to form an immobilized enzyme layer.
 On the surface was applied by spin coating a 0, 0.02, 0.06, 0.1 or 0.3 wt %
 of fluoroalcohol methacrylate resin at 3000 rpm for 30 sec.
 The fluoroalcohol methacrylate resin was Florard 722 (Sumitomo 3M). The
 concentration of the stock solution of Florard 722 was 2 wt % when
 converted to the fluoroalcohol methacrylate resin. Perfluorohexane as a
 diluent was Florard 726 (Sumitomo 3M). The fluoroalcohol methacrylate
 resin was applied as a solution appropriately diluted with Florard 726.
 From the results of relative outputs for each sensor as shown in FIG. 6,
 glucose-permeation restricting property was observed for a fluoroalcohol
 methacrylate resin concentration of 0.02 or higher wt %, 0.1 wt % allowed
 measurement in a wider range, and for 0.3 wt % or higher, current outputs
 were linear to a concentration of 3000 mg/dL which makes it possible to
 measure a higher concentration of glucose solution. An average response
 time for the sensor was below about 15 sec. Such a slow response was
 achieved because the permeation restricting layer could be formed as a
 very thin even film. Spin coating at 3000 rpm for 30 sec as was in this
 example may provide an even film about 0.01 .mu.m to 0.1 .mu.m of
 thickness.
 Example 4
 On a 10 mm.times.6 mm quartz substrate were formed a working electrode of
 platinum (area: 7 mm.sup.2), a counter electrode (area: 4 mm.sup.2) and a
 reference electrode of silver/silver chloride (area: 1 mm.sup.2).
 Then, on the surface was applied by spin coating a 1 v/v% solution of
 y-aminopropyltriethoxysilane to form a binding layer, on which was applied
 by spin coating a 5 w/v% solution of perfluorocarbon-sulfonate polymer, to
 form an ion-exchange polymer layer mainly consisting of the
 perfluorocarbon-sulfonate polymer (NAFION) and then applied by spin
 coating a 22.5 w/v% solution of albumin containing glutaraldehyde at 1
 v/v% to form an immobilized enzyme layer.
 Then, two types of enzyme electrodes were manufactured, with different
 structures of the outermost layer.
 For one enzyme electrode, on the surface of the immobilized enzyme layer
 was applied by spin coating and then dried a 0.3 wt % solution of a
 fluoroalcohol methacylate resin in perfluorohexane at 3000 rpm for 30 sec.
 The fluoroalcohol methacrylate resin was Florard 722 (Sumitomo 3M), whose
 average molecular weight (Mn) of about 7000. Perfluorohexane as a diluent
 was Florard 726 (Sumitomo 3M).
 For the other enzyme electrode, on the surface of the immobilized enzyme
 layer was applied by spin coating and dried a 10 w/v% solution of a
 polyalkylsiloxane in toluene. The polyalkylsiloxane was Pergan Z (Dow
 Corning).
 Two sensors comprising one of these enzyme electrodes thus manufactured
 were soaked for storing in a buffer solution of TES (Methyl
 N-tris(hydroxymethyl)-2-aminoethanesulfonate), pH 7, containing sodium
 chloride at 150 mM, while ten consecutive measurements were conducted for
 a 200 mg/dL standard solution of glucose containing urea at 400 mg/dL.
 FIG. 7 shows outputs of these sensors for the glucose solution as relative
 values where the initial outputs were regarded as 100%.
 As a result, a sensor output was reduced from the second measurement to
 finally 86% in 10th measurement for the sensor comprising a
 polyalkylsiloxane while outputs were stable throughout 10 consecutive
 measurements for the sensor comprising a fluoroalcohol methacrylate resin.
 Such output stability in the sensor comprising a fluoroalcohol
 methacrylate resin can be explained by a lower surface free energy of the
 fluoroalcohol methacrylate resin than the polyalkylsiloxane, by which a
 reduced amount of urea is adhered.
 Example 5
 On a 10 mm.times.6 mm quartz substrate were formed a working electrode of
 platinum (area: 7 mm.sup.2), a counter electrode (area: 4 mm.sup.2) and a
 reference electrode of silver/silver chloride (area: 1 mm.sup.2).
 Then, on the surface was applied by spin coating a 1 v/v% solution of
 y-aminopropyltriethoxysilane to form a binding layer, on which was applied
 by spin coating a 5 w/v% solution of perfluorocarbon-sulfonate polymer, to
 form an ion-exchange polymer layer mainly consisting of the
 perfluorocarbon-sulfonate polymer (NAFION) and then applied by spin
 coating a 22.5 w/v% solution of albumin containing glutaraldehyde at 1
 v/v% to form an immobilized enzyme layer.
 Then, two types of enzyme electrodes were manufactured, with different
 structures of the outermost layer.
 For one enzyme electrode, on the surface of the immobilized enzyme layer
 was applied by spin coating and then dried a 0.3 wt % solution of a
 fluoroalcohol methacylate resin in perfluorohexane at 3000 rpm for 30 sec.
 The fluoroalcohol methacrylate resin was Florard 722 (Sumitomo 3M), whose
 average molecular weight (Mn) of about 7000. Perfluorohexane as a diluent
 was Florard 726 (Sumitomo 3M).
 For the other enzyme electrode, on the surface of the immobilized enzyme
 layer was applied by spin coating and dried a 10 w/v% solution of a
 polyalkylsiloxane in toluene. The polyalkylsiloxane was Pergan Z (Dow
 Corning).
 Two sensors comprising one of these enzyme electrodes thus manufactured
 were soaked for storing in a buffer solution of TES (Methyl
 N-tris(hydroxymethyl)-2-aminoethanesulfonate), pH 7, containing sodium
 chloride at 150 mM, while ten consecutive measurements were conducted for
 a normal urine control for quantification (Lifocheck; Biorad Ltd.)
 containing about 20 mg/dL glucose. FIG. 8 shows outputs of these sensors
 for the glucose solution as relative values where the initial outputs were
 regarded as 100%.
 As a result, a sensor output was reduced from the second measurement to
 finally 28% in 10th measurement for the sensor comprising a
 polyalkylsiloxane while outputs were stable throughout 10 consecutive
 measurements for the sensor comprising a fluoroalcohol methacrylate resin.
 It indicates that the fluoroalcohol methacrylate resin can practically
 adequately minimize adhesion of materials causing sensor output reduction
 in urine.
 Such output stability in the sensor comprising a fluoroalcohol methacrylate
 resin can be explained by a lower surface free energy of the fluoroalcohol
 methacrylate resin than the polyalkylsiloxane, by which the sensor does
 not react with interferent materials causing sensor output reduction such
 as urea.
 Example 6
 On a quartz substrate were formed a counter electrode, a reference
 electrode and three working electrodes (electrode area: 3 mm.sup.2) Then,
 on the overall surface was applied by spin coating a 1 v/v% solution of
 .gamma.-aminopropyltriethoxysilane. Then, on the three working electrodes
 were applied by spin coating a) a 22.5 w/v% solution of albumin containing
 glucose oxidase and glutaraldehyde at 1 v/v%, b) a 22.5 w/v% solution of
 albumin containing lactate oxidase and glutaraldehyde at 0.5 v/v% and c) a
 22.5 w/v% solution of albumin containing ethanol oxidase and
 glutaraldehyde at 1 v/v%.
 After thus forming an immobilized enzyme layer, two types of enzyme
 electrodes were manufactured, with different structures of the outermost
 layer.
 For one enzyme electrode, on the surface of the immobilized enzyme layer
 was applied by spin coating and then dried a 0.3 wt % solution of a
 fluoroalcohol methacylate resin in perfluorohexane at 3000 rpm for 30 sec.
 The fluoroalcohol methacrylate resin was Florard 722 (Sumitomo 3M), whose
 average molecular weight (Mn) of about 7000. Perfluorohexane as a diluent
 was Florard 726 (Sumitomo 3M).
 For the other enzyme electrode, on the immobilized enzyme layer was applied
 by spin coating and dried a 5 w/v% solution of a perfluorocarbon-sulfonate
 polymer to form an ion-exchange polymer layer mainly consisting of the
 perfluorocarbon-sulfonate polymer (NAFION).
 Variation in measurement values was evaluated in repeated measurements
 using the sensors comprising an enzyme electrode. Specifically, 11
 repeated measurements were conducted for a mixed solution containing 100
 mg/dL glucose, 20 mM lactic acid and 20 mM ethanol to determine a
 coefficient of variation (CV value) for the measurement values. A
 coefficient of variation is a value represented by standard
 (deviation/average)X100.
 The sensor comprising an enzyme electrode whose outermost layer consisted
 of a fluoroalcohol methacrylate gave stable measurement values
 irrespective of a measured component as shown in Table 2. It would be
 because the above resin layer does not have electric charge so that it
 little interacts with electrolytic substances such as lactic acid; the
 above resin layer is highly resistant to measured materials such as
 ethanol; or two or more working electrodes and two or more enzymes on the
 same insulating substrate does not mutually affect measurement for each
 target component.
 On the other hand, for the enzyme electrode comprising the outermost layer
 of the perfluorocarbon- sulfonate polymer, the resin film is soluble to
 ethanol, so that its permeation-restricting property is lowered when
 measuring ethanol. In addition, the resin film has electric charge, which
 may cause a larger variation when measuring an electrolytic substance such
 as lactic acid.
 TABLE 2
 Evaluation of a coefficient of variation in measuring different components
 Component Coefficient of variation (%)
 Outermost layer of a fluoroalcohol methacylate resin
 Glucose 3.1
 Lactic acid 3.0
 Ethanol 3.3
 Outermost layer of a perfluorocarbon-sulfonate polymer (NAFION)
 Glucose 2.8
 Lactic acid 6.7
 Ethanol 72
 Example 7
 On a 10 mm.times.6 mm quartz substrate were formed a working electrode of
 platinum (area: 7 mm.sup.2), a counter 10 electrode (area: 4 mm.sup.2) and
 a reference electrode of silver/silver chloride (area: 1 mm.sup.2).
 Then, on the overall surface was applied by spin coating a 1 v/v% solution
 of y-aminopropyltriethoxysilane to form a binding layer, on which was
 applied by spin coating a 22.5 w/v% solution of albumin containing
 glutaraldehyde at 1 v/v%, to form an immobilized enzyme layer.
 Then, the following two types of enzyme electrodes were manufactured, with
 different structures of the outermost layer.
 (1) On the overall surface of the immobilized enzyme layer was applied by
 spin coating and then dried a 0.3 wt % solution of a fluoroalcohol
 methacylate resin in perfluorohexane, to form the first enzyme electrode.
 Spin coating was conducted under the conditions of 3000 rpm and 30 sec.
 The fluoroalcohol methacrylate resin was Florard 722 (Sumitomo 3M),
 1H,1H-perfluorooctyl polymethacrylate with an average molecular weight
 (Mn) of about 7000. Perfluorohexane as a diluent was Florard 726 (Sumitomo
 3M).
 (2) On the overall surface of the immobilized enzyme layer was applied by
 spin coating and dried a 1.7 wt % solution of a copolymer of
 1H,1H,2H,2H-perfluorodecyl polyacrylate and cyclohexyl methacrylate in
 hexafluoroxylene to form the second enzyme electrode, where the
 copolymerization ratio of 1H,1H,2H,2H-perfluorodecyl polyacrylate and
 cyclohexyl methacrylate was about 8:2, i.e., the ratio a/b was about 8/2
 where "a" and "b" represent the numbers of 1H,1H,2H,2H-perfluorodecyl
 acrylate and cyclohexyl methacrylate moieties, respectively.
 Two sensors comprising one of the first and the second enzyme electrodes
 thus manufactured were soaked for storing in a buffer solution of TES
 (Methyl N-tris(hydroxymethyl)-2-aminoethanesulfonate), pH 7, containing
 sodium chloride at 150 mM at 40.degree. C. After 48 hours, 0 to 2000 mg/dL
 standard solutions of glucose were measured with these sensors. An applied
 voltage to the working electrode was 700 mV with reference to the
 reference electrode.
 Thus, stability of a sensor output at 40.degree. C. was tested. FIG. 12
 shows measurement results for a sensor output before and 48 hours after
 soaking. FIGS. 12(a) and 12(b) show measurement results using the sensors
 comprising the first and the second enzyme electrodes, respectively. The
 sensor comprising the first enzyme electrode did not exhibit adequate
 stability while the sensor comprising the second enzyme electrode
 exhibited substantially the same outputs before (0 h in the figures) and
 48 hours after soaking, showing excellent stability.
 Example 8
 On a 10 mm.times.6 mm quartz substrate were formed a working electrode of
 platinum (area: 7 mm.sup.2), a counter electrode (area: 4 mm.sup.2) and a
 reference electrode of silver/silver chloride (area: 1 mM.sup.2).
 Then, on the overall surface was applied by spin coating a 1 v/v% solution
 of .gamma.-aminopropyltriethoxysilane to form a binding layer, on which
 was applied by spin coating a 22.5 w/v% solution of albumin containing
 glutaraldehyde at 1 v/v%, to form an immobilized enzyme layer.
 Then, on the surface was applied by spin coating a solution of a) 85 wt %
 1H,1H,2H,2H-perfluorodecyl polyacrylate and b) 0.085 wt % cyclohexyl
 polymethacrylate in hexafluoroxylene, to form the second enzyme electrode.
 For the sensor comprising the enzyme electrode thus manufactured,
 calibration curves were obtained using 0 to 2000 mg/dL standard solutions
 of glucose. An applied voltage to the working electrode was 700 mV with
 reference to the reference electrode. The calibration curves are shown in
 FIGS. 13(a) and (b), which were obtained for the same sensor. As seen from
 these figures, the sensor of this example exhibited good sensitivity in a
 wide rage of 0 to 2000 mg/dL; in particular it gave an adequate current to
 glucose at 2 to 50 mg/dL. The sensor of this example may be, therefore,
 used to determine an urinary-sugar level for an individual whose
 urinary-sugar level is normal or a prediabetic individual, making it
 possible to collect data useful in prophylaxis for diabetes.
 Ten consecutive measurements were conducted for an urine sample from a
 healthy individual containing glucose at about 2 mg/dL. The results are
 shown in FIG. 14. A reproductivity was about 3% and thus a lower level of
 glucose contained in urine could be determined with a good reproductivity.
 Example 9
 On a 10 mm.times.6 mm quartz substrate were formed a working electrode of
 platinum (area: 7 mm.sup.2), a counter electrode (area: 4 mm.sup.2) and a
 reference electrode of silver/silver chloride (area: 1 mm.sup.2). The
 substrate was soaked in a 6M solution of urea containing sodium chloride
 at 150 mM, while a voltage of 0.7 V to the working electrode with
 reference to the reference electrode was applied for 10 min, to form an
 urea layer as an electrode protection layer on the working electrode.
 Formation of the urea layer was confirmed by observing infrared absorption
 spectra for a sample treated in a similar manner. Then, infrared
 absorption signals from urea were observed at 3440, 3340, 1640 and 1470
 cm.sup.-1. Since urea is more adhesive to platinum, the urea layer was
 selectively formed on the platinum electrode.
 Then, on the surface was applied by spin coating a 1 v/v% solution of
 .gamma.-aminopropyltriethoxysilane to form a binding layer, on which was
 applied by spin coating a 22.5 w/v% solution of albumin containing
 glutaraldehyde at 1 v/v%, to form an immobilized enzyme layer for
 providing an enzyme electrode.
 As a control, an enzyme electrode was manufactured as described above
 except that an urea layer was not formed.
 Two glucose sensors comprising one of these electrodes (detection unit)
 thus manufactured were soaked for storing in a buffer solution of TES
 (Methyl N-tris(hydroxymethyl)-2-aminoethanesulfonate), pH 7, containing
 sodium chloride at 150 mM, while five to ten consecutive measurements were
 conducted for a normal urine control for quantification (Lifocheck; Biorad
 Ltd.) containing about 20 mg/dL glucose. FIG. 20 shows outputs of these
 sensors for the glucose solution as relative values where the initial
 outputs were regarded as 100%.
 As a result, a sensor output was sharply reduced from the second
 measurement to finally 52% in fourth measurement for the sensor without an
 urea layer while outputs were stable throughout 10 consecutive
 measurements for the sensor comprising an urea layer. The sensor
 comprising an urea layer gave such stable outputs because the film
 substantially completely restricted permeation of the contaminants in the
 normal urine control for quantification to the electrode.
 Example 10
 On a 10 mm.times.6 mm quartz substrate were formed a working electrode of
 platinum (area: 7 mm.sup.2), a counter electrode (area: 4 mm.sup.2) and a
 reference electrode of silver/silver chloride (area: 1 mm.sup.2). The
 substrate was soaked in a 6M solution of urea containing sodium chloride
 at 150 mM, while a voltage of 0.7 V at the working electrode with
 reference to the reference electrode was applied for 10 min, to form an
 urea layer as an electrode protection layer on the working electrode.
 Then, on the surface was applied by spin coating a 1 v/v% solution of
 .gamma.-aminopropyltriethoxysilane to form a binding layer, on which was
 applied by spin coating a 5 w/v% solution of a perfluorocarbon-sulfonate
 polymer (NAFION), to form an ion-exchange polymer layer.
 As a control, a binding layer and an ion-exchange layer were formed on the
 electrode as described above except that an urea layer was not formed.
 Two glucose sensors comprising one of these electrodes (detection unit)
 thus manufactured were soaked for storing in a buffer solution of TES
 (Methyl N-tris(hydroxymethyl)-2-aminoethanesulfonate), pH 7, containing
 sodium chloride at 150 mM, while sensor outputs were determined for 100
 .mu.M hydrogen peroxide, 50 mg/dL ascorbic acid and 8 mg/dL acetaminophen.
 Outputs from the sensor with an urea layer are shown as relative values
 where an output from the sensor without an urea layer is regarded as 100%
 (FIG. 21).
 It was consequently indicated that the urea layer formed on the surface of
 the working electrode restricted permeation of ascorbic acid and
 acetaminophen as interferent substances to improve selective permeability
 for hydrogen peroxide.
 Example 11
 On a 10 mm.times.6 mm quartz substrate were formed a working electrode of
 platinum (area: 7 mm.sup.2), a counter electrode (area: 4 mm.sup.2) and a
 reference electrode of silver/silver chloride (area: 1 mm.sup.2). The
 substrate was soaked in a 6M solution of urea containing sodium chloride
 at 150 mM, while a voltage of 0.7 V at the working electrode with
 reference to the reference electrode was applied for 1, 3, 9 or 27 min,
 i.e., an urea layer was formed, varying a voltage application time.
 Then, on the surface was applied by spin coating a 1 v/v% solution of
 y-aminopropyltriethoxysilane to form a binding layer, on which was applied
 by spin coating a 5 w/v% solution of a perfluorocarbon-sulfonate polymer
 (NAFION), to form an ion-exchange polymer layer.
 As a control, a binding layer and an ion-exchange layer were formed on the
 electrode as described above except that an urea layer was not formed.
 Two glucose sensors comprising one of these electrodes (detection unit)
 thus manufactured were soaked for storing in a buffer solution of TES
 (Methyl N-tris(hydroxymethyl)-2-aminoethanesulfonate), pH 7, containing
 sodium chloride at 150 mM, while sensor outputs were determined for 100
 .mu.M hydrogen peroxide, 50 mg/dL ascorbic acid and 8 mg/dL acetaminophen.
 Output from each sensor with a given voltage application time are shown as
 relative values where an output from the sensor without an urea layer is
 regarded as 100% (FIG. 22).
 It was consequently indicated that an urea layer formed on the sensor by
 applying the voltage for at least 3 min effectively restricted permeation
 of ascorbic acid and acetaminophen as interferent substances to improve
 selective permeability for hydrogen peroxide.
 Example 12
 On a 10 mm.times.6 mm quartz substrate were formed a working electrode of
 platinum (area: 7 mm.sup.2), a counter electrode (area: 4 mm.sup.2) and a
 reference electrode of silver/silver chloride (area: 1 mm.sup.2). The
 substrate was soaked in a 0.1 mM, 0.1 M, 1 M or 6 M solution of urea
 containing sodium chloride at 150 nM, while a voltage of 700 mV at the
 working electrode with reference to the reference electrode was applied
 for 10 min.
 Then, on the surface was applied by spin coating a 1 v/v% solution of
 .gamma.-aminopropyltriethoxysilane to form a binding layer, on which was
 applied by spin coating a 5 w/v% solution of a perfluorocarbon-sulfonate
 polymer (NAFION), to form an ion-exchange polymer layer.
 As a control, a binding layer and an ion-exchange layer were formed on the
 electrode as described above except that an urea layer was not formed.
 Two glucose sensors comprising one of these electrodes (detection unit)
 thus manufactured were soaked for storing in a buffer s olution of TES
 (Methyl N- tris(hydroxymethyl)-2-aminoethanesulfonate), pH 7, containing
 sodium chloride at 150 mM, while sensor outputs were determined for 100
 .mu.M hydrogen peroxide, 50 mg/dL ascorbic acid and 8 mg/dL acetaminophen.
 Output from each sensor with a given voltage application time are shown as
 relative values where an output from the sensor without an urea layer is
 regarded as 100% (FIG. 23).
 It was consequently indicated that treating the electrode with at least 0.1
 mM urea solution at an applied voltage of 700 mV for 10 min provided good
 selectivity for hydrogen peroxide.
 Example 13
 On a 10 mm.times.6 mm quartz substrate were formed a working electrode of
 platinum (area: 7 mm.sup.2), a counter electrode (area: 4 mm.sup.2) and a
 reference electrode of silver/silver chloride (area: 1 mm.sup.2). The
 substrate was soaked in a 6M solution of urea containing sodium chloride
 at 150 mM, while a voltage of 0.7 V to the working electrode with
 reference to the reference electrode was applied for 10 min, to form an
 urea layer on the working electrode. Formation of the urea layer was
 confirmed by observing infrared absorption spectra for a sample treated in
 a similar manner.
 Then, on the surface was applied by spin coating a 1 v/v% solution of
 .gamma.-aminopropyltriethoxysilane to form a binding layer, on which were
 sequentially applied by spin coating a 5 w/v% solution of a
 perfluorocarbon-sulfonate polymer (NAFION) to form an ion-exchange polymer
 layer; a 22.5 w/v% solution of albumin containing glucose oxidase and
 glutaraldehyde at 1 v/v%, to form an immobilized enzyme layer; and a 0.3
 wt % solution of a polyfluoroalcohol methacrylate resin in perfluorohexane
 to form a permeation restricting layer, for providing an enzyme electrode.
 As a control, an enzyme electrode was manufactured as described above
 except that an urea layer was not formed.
 Two glucose sensors comprising one of these electrodes (detection unit)
 thus manufactured were soaked for storing in a buffer solution of TES
 (Methyl N-tris(hydroxymethyl)-2-aminoethanesulfonate), pH 7, containing
 sodium chloride at 150 mM, while four to six consecutive measurements were
 conducted for an abnormal urine control for quantification (Lifocheck;
 Biorad Ltd.) containing about 302 mg/dL glucose. FIG. 24 shows outputs of
 these sensors for the glucose solution as relative values where the
 initial outputs were regarded as 100%.
 As a result, a sensor output was gradually reduced from the second
 measurement to finally 53% in fourth measurement for the sensor without an
 urea layer while outputs were stable throughout 6 consecutive measurements
 for the sensor comprising an urea layer. The sensor comprising an urea
 layer gave such stable outputs because the film substantially completely
 restricted permeation of the contaminants in the abnormal urine control
 for quantification to the electrode.
 Example 14
 On a 10 mm.times.6 mm quartz substrate were formed a working electrode of
 platinum (area: 7 mm.sup.2), a counter electrode (area: 4 mm.sup.2) and a
 reference electrode of silver/silver chloride (area: 1 mm.sup.2). The
 substrate was soaked in a 6M solution of urea containing sodium chloride
 at 150 mM, while a voltage of 0.7 V to the working electrode with
 reference to the reference electrode was applied for 10 min, to form an
 urea layer on the working electrode.
 Then, on the surface was applied by spin coating a 1 v/v% solution of
 .gamma.-aminopropyltriethoxysilane to form a binding layer, on which were
 applied by spin coating a 5 w/v% solution of a perfluorocarbon-sulfonate
 polymer (NAFION) to form an ion-exchange polymer layer for providing a
 detection unit of a sensor.
 As a control, on a 10 mm.times.6 mm quartz substrate were formed a working
 electrode of platinum (area: 7 mm.sup.2), a counter electrode (area: 4
 mm.sup.2) and a reference electrode of silver/silver chloride (area: 1
 mm.sup.2). On the surface were sequentially applied by spin coating a 1
 v/v% solution of .gamma.-aminopropyltriethoxysilane, a 2 w/v% solution of
 acetylcellulose ad a 5 w/v% solution of a perfluorocarbon-sulfonate
 polymer (NAFION), to form an electrode unit of a sensor.
 Two glucose sensors comprising one of these electrodes (detection unit)
 thus manufactured were soaked for storing in a buffer solution of TES
 (Methyl N-tris(hydroxymethyl)-2-aminoethanesulfonate), pH 7, containing
 sodium chloride at 150 mM, while sensor outputs were determined for 100
 .mu. M hydrogen peroxide, 50 mg/dL ascorbic acid and 8 mg/dL acetaminophen
 (FIG. 25).
 Consequently, the sensor comprising an urea layer could selectively measure
 hydrogen peroxide at least 36 consecutive days, while the sensor
 comprising the acetylcellulose layer could measure it only for 3 days and
 subsequent measurements could not be conducted due to failure of the
 sensor. The acetylcellulose layer could not be formed as an even film, so
 that the layer was detached during operation, resulting in the failure of
 the sensor itself.
 Example 15
 This example relates to an example of a measuring apparatus having the
 configuration shown in FIG. 31.
 First, there will be described a procedure for manufacturing a biosensor
 unit in the measuring apparatus of this example. On a 10 mm.times.6 mm
 quartz substrate were formed a working electrode of platinum (area: 7
 mm.sup.2), a counter electrode (area: 4 mm.sup.2) and a reference
 electrode of silver/silver chloride (area: 1 mm.sup.2). Then, on the
 overall surface were sequentially applied by spin coating a 1 v/v%
 solution of y-aminopropyltriethoxysilane to form a binding layer; a 22.5
 w/v% solution of albumin containing 56.5 U/.mu.L glucose oxidase and 1
 v/v% glutaraldehyde to form an immobilized enzyme layer; and a 1.7 wt %
 solution of a polyfluoroalcohol acrylate resin to form a permeation
 restricting layer. The polyfluoroalcohol acrylate resin was
 1H,1H,2H,2H-perfluorodecyl polyacrylate. The diluent was hexafluoroxylene.
 The conditions of spin coating were 3000 rpm and 30 sec.
 Using a biosensor comprising an electrode unit thus manufactured, a
 measuring apparatus having a configuration shown in FIG. 31 was
 manufactured, where the electrode unit was connected, via wire bonding,
 with a flexible substrate, which was connected with an electrochemical
 measuring circuit via a pin-jack type wiring.
 The electrochemical measuring circuit was a potentiostat,
 HOKUTODENKOPOTENTIOSTAT/GALVANOSTATHA150G (Hokuto Denko). The data
 processor was a personal computer, PC-9821RaII23 (NEC corporation). The
 data indicator 53 was a display, PC-KP531 (NEC corporation). The
 electrochemical measuring circuit, the data processor and the data
 indicator 53 were mutually connected via a pin-jack type wiring.
 Operation of the measuring apparatus of this example will be described.
 An operator soaked the biosensor comprising an enzyme electrode into a
 buffer of 1 mM TES (Methyl N-tris(hydroxymethyl)-2-aminoethanesulfonate),
 pH 7, containing sodium chloride at 150 mM, and turned the apparatus on.
 Then, the data indicator displayed a message "Time setting; Enter a
 current time". According to the indication, the operator entered the
 current time by key operation, and then the data indicator displayed the
 message "A current time was entered". When an entered time was incorrect,
 the message "Time setting; Enter a current time" is displayed. Thus, the
 entered current time is stored in the data processor.
 Then, the data indicator displayed a message "Standby mode. Please wait.".
 After a current from the enzyme electrode, the data indicator displayed a
 message "Calibration; Soak the electrode into a calibration solution".
 According to the indication, the operator soaked the biosensor into a
 calibration solution of 200 mg/dL glucose for calibration. Then, the data
 indicator displayed a message "Calibration was normally finished. After
 washing, resoak the electrode into the buffer.". Whether the calibration
 has been normally conducted is judged by the data processor and the result
 is displayed on the data indicator. If the calibration is not normally
 conducted, a message "Not calibrated. After washing the electrode, resoak
 it into the calibration solution" or "The electrode is broken. Replace
 it." is displayed. After the calibration, the operator soaked the enzyme
 electrode into the buffer for measurement. When the electrode is not
 returned into the buffer 10 sec after the calibration is completed, an
 alarm sounds.
 Then, the operator selected the item "Start measurement" displayed on the
 data indicator, and then the indicator displayed a message "Measurement
 will be started. Soak the electrode into an urine sample.". According the
 indication, the operator soaked the electrode into an urine sample to
 initiate measurement. Ten seconds after the measurement initiation, the
 data indicator displayed a message "Measurement has been normally
 finished. An urinary-sugar level is . . . mg/dL.". Whether the measurement
 has been normally conducted is judged by the data processor and the result
 is displayed on the data indicator. If the measurement is not normally
 conducted, a message "Not measured. After washing the electrode, resoak it
 into the urine solution" or "The electrode is broken. Replace it." is
 displayed.
 After the measurement, the data indicator displayed a message "Wash and
 resoak the electrode into the buffer". When the electrode is not returned
 into the buffer 10 sec after the measurement is completed, an alarm
 sounds. Then, the operator selected the item "Completion of measurement"
 on the data indicator, to complete the measurement.
 For the measuring apparatus of this example, a measuring time may be set in
 advance. At the set time, an indicating sound generates while the data
 indicator displays a message "Measurement will be started. Soak the
 electrode into an urine sample.". The time may be set as appropriate and a
 plurality of times may be set.
 After entering data in the data processor in the measuring apparatus of
 this invention, an indicating sound is generated in either case that the
 entry is acceptable or unacceptable. An indicating light rather than an
 indicating sound may be employed. When an abnormal current is detected
 between the enzyme electrode, the electrochemical measuring circuit, the
 data processor and the wiring, an abnormal-current indicator displays a
 message "lAn abnormal current was detected" on the data indicator.
 Displaying the message may prevent failure of the apparatus.
 Since all of the biosensor, the electrochemical measuring circuit and the
 data processor are connected via a pin-jack type wiring, they are readily
 removed and may be replaced as necessary.
 As described above, the measuring apparatus of this example may be used to
 regularly conduct measurement at a given time without misoperation and
 anyone can easily operate it.
 Example 16
 This example relates to an example of a measuring apparatus having a
 configuration shown in FIG. 33. The measuring apparatus is the apparatus
 as described in Example 15 further comprising a pH sensor and a
 temperature sensor.
 The temperature sensor was a thermocouple type and the pH sensor was an
 ion-sensitive field effect transistor type. The pH sensor, the temperature
 sensor, the electrochemical measuring circuit, the data processor and the
 data indicator were mutually connected via an electric wire.
 Operation of the measuring apparatus of this example will be described.
 An operator soaked the enzyme electrode into a buffer of 1 nM TES (Methyl
 N-tris(hydroxymethyl)-2-aminoethanesulfonate), pH 7, containing sodium
 chloride at 150 mM, and turned the apparatus on. After 1 min, the base
 current of the enzyme electrode became stable. In this state, the enzyme
 electrode was soaked into a 200 mg/dL standard solution of glucose for
 calibration. Since the glucose standard solution contains a pH indicator,
 the pH sensor can be calibrated at the same time. Except replacement of
 the electrode, the apparatus may be kept power-ON as long as the enzyme
 electrode is connected.
 Then, the operator selected the item "Start measurement", and the data
 indicator displayed a message "Measurement will be started. Soak the
 electrode into an urine sample.".
 According the indication, the operator sequentially measured an
 urinary-sugar level for samples from two diabetic subjects once per a
 sample. During the measurement, a memo registration means was used to
 enter a blood pressure and a temperature at the same time for each
 subject. Then, 10 sec. after the first subject soaked the electrode into
 urine, the data indicator displayed a message "Measurement has been
 normally finished. An urinary-sugar level is 80 mg/dL." and a voice
 message "Measurement has been normally finished. An urinary-sugar level is
 80 mg/dL." sounded. After 20 sec, the second subject soaked the enzyme
 electrode into urine. After 10 sec, the data indicator displayed a message
 "Measurement has been normally finished. An urinary-sugar level is 180
 mg/dL." and a voice message "Measurement has been normally finished. An
 urinary-sugar level is 180 mg/dL." sounded. These results were compared
 with those obtained from an existing laboratory apparatus (Hitachi
 Automatic Analyzer 7050 according to a glucose dehydrogenase technique).
 There were good matching and a higher correlation between the
 measurements.
 Thus, the measuring apparatus of this example may be used to sequentially
 detect an urinary-sugar level for two subjects. Even a subject with a weak
 eyesight could reliably determine his/her urinary-sugar level. In
 addition, the memo-registration means could be used to access a
 temperature and a blood pressure entered in advance. It allowed an
 operator to compare these values to the urinary-sugar level for carefully
 administrating subject's conditions. Furthermore, the measured values were
 corrected for a temperature and a pH, so that highly precise detection
 comparable to an existing laboratory apparatus could be conducted.
 Example 17
 This example relates to an example of a measuring apparatus having a
 configuration shown in FIG. 34. This measuring apparatus is the apparatus
 in Example 16 further comprising a communication processor 58.
 The communication processor 58 was a modem-terminal adapter, PC-IT65S1P
 (NEC corporation). The pH sensor, the temperature sensor, the
 electrochemical measuring circuit, the data processor, the data indicator
 and the communication processor were mutually connected via an electric
 wire.
 This apparatus was used to detect an urinary-sugar level for one diabetic
 twice (2 hours after breakfast and dinner) a day for 30 days. A measured
 value was sent to a hospital via a telephone line one by one.
 Thus, the patient successfully observed the measuring times, so that the
 hospital could plot the received data into a graph and analyze it for
 appropriately administrating the patient's conditions.
 Example 18
 This example relates to an example of a measuring apparatus having a
 configuration shown in FIG. 35. The measuring apparatus is the apparatus
 in Example 17 further comprising a printer 59. The printer 59 was a laser
 printer, Multiwriter 2000X (NEC corporation). The printer was connected
 with the data processor via a printer cable, PC-CA202. There will be
 described measurement results using the measuring apparatus of this
 example.
 This apparatus was used to detect an urinary-sugar level for consecutive
 100 diabetics. The apparatus was calibrated only once when starting it.
 For the same samples, an urinary-sugar level was detected with an existing
 laboratory apparatus (Hitachi Automatic Analyzer 7050 according to a
 glucose dehydrogenase technique), whose results were compared with those
 from the measuring apparatus of this example. Then, a coefficient of
 correlation obtained was 0.96 and a regression formula was Y=1.09X+88.
 It-was shown that the apparatus of this example could conduct detection
 with a precision comparable to that in the existing laboratory apparatus.
 A measuring time for the apparatus of this example was as short as about
 90 sec per a sample, allowing an operator to quickly conduct detection.
 Furthermore, the apparatus of this example was equipped with the printer
 59, so that the measurement results could be quickly printed for
 confirmation. The patient could show a physician the printed results in a
 hospital for receiving his/her advice.
 Example 19
 This example relates to an example of a measuring apparatus having a
 configuration shown in FIG. 36. The measuring apparatus is the apparatus
 as described in Example 18 further comprising an external storage 55. The
 external storage was a 3.5 inch optical disk unit, PC-OD302R (NEC
 corporation). The external storage was connected with the data processor
 via an electric wire. Operation of the measuring apparatus of this example
 will be described.
 An operator soaked the enzyme electrode into a buffer of 1 mM TES (Methyl
 N-tris(hydroxymethyl)-2-aminoethanesulfonate), pH 7, containing sodium
 chloride at 150 mM, and turned the apparatus on. After about 1 min, the
 base current of the enzyme electrode became stable. In this state, the
 enzyme electrode was soaked into a 200 mg/dL standard solution of glucose
 for calibration and the enzyme electrode was calibrated.
 Then, the operator selected an item "Entry of the number of subjects"
 displayed on the data indicator, and a message "Enter the number of
 subjects" was displayed. After the operator entered the number of subjects
 according to this instruction, the data indicator displayed a message ". .
 . subjects will be tested. (Yes, Y/No. N)". "Yes, Y" was selected and then
 a message ". . . subjects can be tested" was displayed. If "No, N" is
 selected, the message "Enter the number of subjects" is again displayed
 and the above procedure is repeated until "Yes, Y" is selected.
 The operator selected an item "Password" on the data indicator and then
 "Registration of a password". The data processor recognizes that the
 password entering button has been pushed to make the data indicator
 display a message "A password will be registered. Enter a 4 digit
 number.". After the operator entered a 4 digit number according to this
 instruction, the data indicator displayed a message "Enter the same
 password". After the operator entered the same number again, a message
 "The password was received" was displayed. Passwords are registered by the
 number of subjects. Thus, the registered passwords are stored in the
 memory in the data processor.
 Then, the data indicator displayed a message "Measurement will be started.
 After entering the password, soak the electrode into an urine sample.".
 According to this instruction, the operator entered the password and soak
 the electrode into urine to start detection. Then, the data indicator
 displayed a message "Measurement has been normally finished. An
 urinary-sugar level is . . . mg/dL.". If the measurement is not normally
 conducted, a message "Not measured. After washing the electrode, resoak it
 into the urine solution" or "The electrode is broken. Replace it." is
 displayed. If a correct password is not entered, the message "Measurement
 will be started. After entering the password, soak the electrode into an
 urine sample." is again displayed. If an incorrect password is entered
 three consecutive times, all the measured data are deleted and the setting
 returns to the initial state.
 After normally completing detection, the data indicator displayed a message
 "Wash and resoak the electrode into the buffer".
 The operator selected "Memo registration" on the data indicator and then a
 message "A memo is registered? (Yes, Y/No, N)" was displayed. After
 selecting "Y", a message "Enter the password" was displayed. The operator
 entered the password and memo data, and then the data indicator displayed
 the message "A memo is registered? (Yes, Y/No, N)". The operator selected
 "Y" and entered the memo data, and then the data indicator again displayed
 the message "A memo is registered? (Yes, Y/No, N)". The operator selected
 "Y" and registered the memo. For stopping the input process, "N" is
 selected. When reading, amending or deleting a registered memo, a message
 "After entering the password, designate the memo number." is displayed.
 According to the instruction, the operator can enter the password to read,
 amend or delete the memo. If an incorrect password is entered, a message
 "The password is incorrect. Enter the password again." is displayed. If an
 incorrect password is entered three consecutive times, all the memo data
 are deleted and the setting returns to the initial state.
 Measurement results obtained using the measuring apparatus of this example
 will be described. The apparatus of this example was used to repeatedly
 measure an urinary-sugar level for two diabetics twice a day for a week.
 The apparatus was calibrated only once when starting it. For the same
 samples, an urinary-sugar level was detected with an existing laboratory
 apparatus (Hitachi Automatic Analyzer 7050 according to a glucose
 dehydrogenase technique), whose results were compared with those from the
 measuring apparatus of this example. Then, a coefficient of correlation
 obtained was 0.955 (n=28). It was shown that the apparatus of this example
 could conduct detection with a precision comparable to that in the
 existing laboratory apparatus. Since the memo function was used to enter
 the names of the patients, the measurement data were not mixed up. Since
 passwords were used for data management, patient's privacy could be
 protected during measurement. The measurement data could be graphically
 represented. Furthermore, the optical disk storing the data was portable,
 so that the data could be managed or analyzed by another PC.