JPH11344460A - Method for evaluating liquid and culture medium - Google Patents
Method for evaluating liquid and culture mediumInfo
- Publication number
- JPH11344460A JPH11344460A JP10153141A JP15314198A JPH11344460A JP H11344460 A JPH11344460 A JP H11344460A JP 10153141 A JP10153141 A JP 10153141A JP 15314198 A JP15314198 A JP 15314198A JP H11344460 A JPH11344460 A JP H11344460A
- Authority
- JP
- Japan
- Prior art keywords
- electrode
- glucose
- enzyme
- lactic acid
- solution
- Prior art date
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、被検試料液中の二
つの特定成分を同時に定量して、迅速かつ高精度に被検
試料液を評価する方法に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for quantifying two specific components in a test sample solution at the same time to quickly and accurately evaluate the test sample solution.
【０００２】[0002]
【従来の技術】従来、液体中の特定成分を評価する方法
として、ガスクロマトグラフィーや液体クロマトグラフ
ィーを利用する方法、または酵素を利用する方法が知ら
れている。酵素を利用する方法には、酵素反応に伴って
変化する色素を反応系に共存させ、この色素を発色させ
て分光学的に測定する方法と、酵素反応に伴って還元さ
れる物質を反応系に含ませ、その還元された物質の量を
電気化学的に測定する方法がある。2. Description of the Related Art Conventionally, as a method for evaluating a specific component in a liquid, a method utilizing gas chromatography or liquid chromatography or a method utilizing an enzyme is known. Enzyme-based methods include a method in which a dye that changes with the enzymatic reaction coexists in the reaction system, coloration of this dye and spectroscopic measurement, and a method in which the substance that is reduced in the enzymatic reaction is measured in the reaction system. And measuring the amount of the reduced substance electrochemically.
【０００３】例えば、酵素と発色試薬を用いて試料液中
のグルコースを測定する方法について、和光純薬工業
（株）から「グルコースＢ−テストワコー」の商品名で
販売されているセットを用いて説明する。まず、発色試
薬液として、グルコースオキシダーゼ、ペルオキシダー
ゼおよび４−アミノアンチピリンを含む発色剤を緩衝液
に溶解させたものを調製する。次に、２種類のグルコー
ス標準液（２００ｍｇ／ｄｌおよび５００ｍｇ／ｄｌ）
を適当に希釈して、５から６種類の濃度のグルコース溶
液を調製する。[0003] For example, a method for measuring glucose in a sample solution using an enzyme and a coloring reagent is described by using a set sold under the trade name of "glucose B-test Wako" by Wako Pure Chemical Industries, Ltd. explain. First, a color reagent containing glucose oxidase, peroxidase, and 4-aminoantipyrine dissolved in a buffer is prepared as a color reagent solution. Next, two kinds of glucose standard solutions (200 mg / dl and 500 mg / dl)
Is appropriately diluted to prepare glucose solutions of 5 to 6 concentrations.
【０００４】そして、調製したグルコース溶液０．０２
ｍｌに、発色試薬液３．０ｍｌをよく混合し、３７℃で
２０分間加温した後、この混合溶液の５０５ｎｍにおけ
る吸光度をそれぞれ測定する。同様にして、種々の濃度
のグルコース溶液に対する吸光度を測定し、横軸にグル
コース濃度、縦軸に吸光度をプロットして検量線を得
る。被検試料液の測定は、グルコース溶液の代わりに、
被検試料液０．０２ｍｌを用い、上記と同様にして、５
０５ｎｍにおける吸光度を測定する。そして、先に求め
た検量線から、被検試料液中のグルコース濃度を算出す
る。[0004] The prepared glucose solution 0.02
Then, 3.0 ml of the coloring reagent solution is mixed well with the resulting mixture, and the mixture is heated at 37 ° C. for 20 minutes. Then, the absorbance of the mixed solution at 505 nm is measured. Similarly, the absorbances of various concentrations of glucose solutions are measured, and the calibration curve is obtained by plotting the glucose concentration on the horizontal axis and the absorbance on the vertical axis. The measurement of the test sample solution is performed in place of the glucose solution.
Using 0.02 ml of the test sample solution, 5
The absorbance at 05 nm is measured. Then, the glucose concentration in the test sample solution is calculated from the previously obtained calibration curve.
【０００５】また、酵素と発色試薬を用いて試料液中の
Ｌ−乳酸を測定する方法について、ベーリンガー・マン
ハイム（株）から「Ｆ−キット Ｌ−乳酸製」の商品名
で販売されているセットを用いて説明する。まず、Ｌ−
グルタミン酸の緩衝溶液１．００ｍｌ、ニコチンアミド
−アデニンジヌクレオチド溶液０．２０ｍｌ、蒸留水
１．００ｍｌおよびグルタミン酸−ピルビン酸アミノ基
転移酵素溶液０．０２ｍｌを混合して混合溶液を調製す
る。そして、混合５分後の３４０ｎｍにおける吸光度を
測定する。続いて、Ｌ−乳酸脱水素酵素溶液０．０２ｍ
ｌを加え、２０分間反応させた後、吸光度を測定する。
このとき得られた吸光度と最初に測定した吸光度の差を
求めブランク値とする。A method for measuring L-lactic acid in a sample solution using an enzyme and a coloring reagent is disclosed in a set sold by Boehringer Mannheim under the trade name of "F-kit L-lactic acid". This will be described with reference to FIG. First, L-
A mixed solution is prepared by mixing 1.00 ml of a glutamic acid buffer solution, 0.20 ml of a nicotinamide-adenine dinucleotide solution, 1.00 ml of distilled water and 0.02 ml of a glutamic acid-pyruvate aminotransferase solution. Then, the absorbance at 340 nm after 5 minutes of mixing is measured. Then, L-lactic acid dehydrogenase solution 0.02m
After adding l and reacting for 20 minutes, the absorbance is measured.
The difference between the absorbance obtained at this time and the absorbance measured first is determined as a blank value.
【０００６】被検試料液の測定は、まず上記と同様にし
て混合溶液を調製し、この混合溶液に被検試料液０．１
０ｍｌを加えて混合する。そして、混合５分後の３４０
ｎｍにおける吸光度を測定する。続いて、Ｌ−乳酸脱水
素酵素溶液０．０２ｍｌを加え、２０分後の吸光度を測
定する。このとき得られた吸光度と、先に求めたブラン
ク値、および吸光度係数などの定数からＬ−乳酸の濃度
を測定する。For the measurement of the test sample solution, first, a mixed solution is prepared in the same manner as described above, and the test sample solution is added to the mixed solution.
Add 0 ml and mix. And 340 after 5 minutes of mixing
Measure the absorbance in nm. Subsequently, 0.02 ml of L-lactic acid dehydrogenase solution is added, and the absorbance after 20 minutes is measured. The concentration of L-lactic acid is measured from the absorbance obtained at this time, the previously obtained blank value, and constants such as the absorbance coefficient.
【０００７】また、電気化学的な方法で試料液中に特定
成分を測定するには、例えば、特公平７−１１４７０５
号公報に開示されているバイオセンサを用いる方法があ
る。図５は、このバイオセンサの一例を示す平面図であ
る。また、図６は、図５の縦断面図である。絶縁性の基
板１上にスクリーン印刷等の方法で測定極１９、対極２
０および参照極２１からなる電極系を形成する。次に、
絶縁層８を形成して各電極の露出面積を一定にした後
に、上記電極系上に親水性高分子と酸化還元酵素と電子
受容体からなる反応試薬層２２を形成したものである。In order to measure a specific component in a sample solution by an electrochemical method, for example, Japanese Patent Publication No. 7-114705
There is a method using a biosensor disclosed in Japanese Unexamined Patent Publication (Kokai) Publication. FIG. 5 is a plan view showing an example of this biosensor. FIG. 6 is a longitudinal sectional view of FIG. The measuring electrode 19 and the counter electrode 2 are formed on the insulating substrate 1 by screen printing or the like.
An electrode system composed of 0 and the reference electrode 21 is formed. next,
After the insulating layer 8 is formed and the exposed area of each electrode is made constant, a reaction reagent layer 22 composed of a hydrophilic polymer, an oxidoreductase and an electron acceptor is formed on the electrode system.
【０００８】このようにして作製されたバイオセンサの
反応試薬層上に、基質を含む試料液を滴下すると、反応
試薬層が溶解して酵素と基質が反応し、これに伴い電子
受容体が還元される。酵素反応終了後、この還元された
電子受容体を電気化学的に酸化し、このとき得られる酸
化電流値と予め測定装置に記憶されている検量線に基づ
いて被検試料液中の基質濃度が算出される。When a sample solution containing a substrate is dropped on the reaction reagent layer of the biosensor thus prepared, the reaction reagent layer dissolves, and the enzyme and the substrate react with each other, whereby the electron acceptor is reduced. Is done. After the enzymatic reaction, the reduced electron acceptor is electrochemically oxidized, and the substrate concentration in the test sample solution is reduced based on the oxidation current value obtained at this time and the calibration curve stored in the measuring device in advance. Is calculated.
【０００９】[0009]
【発明が解決しようとする課題】ガスクロマトグラフィ
ー、液体クロマトグラフィーおよび発色試薬を用いる酵
素法によって被検試料液中の特定成分を定量するには、
測定する試料液の量を正確にする必要があり、また、測
定時間には少なくとも数分から数十分要するため、簡便
性に欠けていた。さらに、いずれの測定方法においても
高価な測定装置が必要であった。また、培地の特定成分
を測定するには、濾過、除菌といった前処理が必要であ
り、操作が煩雑であった。In order to quantify a specific component in a test sample solution by gas chromatography, liquid chromatography, and an enzymatic method using a coloring reagent,
Since the amount of the sample solution to be measured needs to be accurate, and the measuring time requires at least several minutes to several tens of minutes, it lacks simplicity. Furthermore, an expensive measuring device was required in any of the measuring methods. In addition, in order to measure a specific component of the medium, pretreatment such as filtration and sterilization was required, and the operation was complicated.
【００１０】バイオセンサを用いる場合は、簡易な操作
で高精度な測定が可能であるが、複数の特性成分の濃度
を同時に測定するには、それぞれを測定するバイオセン
サが必要となり、手技が煩雑であった。本発明は、上記
課題を鑑み、被検試料液中の複数の成分、特にグルコー
スと乳酸を含む溶液を簡易な操作で迅速に定量して、被
検試料液を評価する方法を提供することを目的とする。[0010] When a biosensor is used, high-precision measurement can be performed by a simple operation. However, simultaneous measurement of the concentrations of a plurality of characteristic components requires a biosensor for measuring each component, which makes the procedure complicated. Met. The present invention has been made in view of the above problems, and provides a method for quickly quantifying a plurality of components in a test sample solution, particularly a solution containing glucose and lactic acid, with a simple operation to evaluate the test sample solution. Aim.
【００１１】[0011]
【課題を解決するための手段】本発明による液体の評価
法は、絶縁性の基板、前記基板の同一面上または異なる
面上に形成された２組の電極系、および前記２組の電極
系上にそれぞれ形成された２つの反応試薬層からなり、
前記反応試薬層の一方が少なくともグルコース酸化能を
有する酵素を含み、前記反応試薬層の他方が少なくとも
乳酸酸化能を有する酵素を含むバイオセンサを用い、被
検試料液を前記２つの反応試薬層に供給して前記被検試
料液と前記酵素を電子受容体の存在下で反応させる工
程、および前記酵素反応に伴って還元された電子受容体
を電気化学的に測定する工程を含み、被検試料液中グル
コースおよび乳酸をそれぞれ定量することを特徴とす
る。A method for evaluating a liquid according to the present invention comprises an insulating substrate, two sets of electrode systems formed on the same or different surfaces of the substrate, and the two sets of electrode systems. It consists of two reaction reagent layers formed on each,
One of the reaction reagent layers contains an enzyme having at least glucose oxidizing ability, and the other of the reaction reagent layers uses a biosensor containing at least an enzyme having lactic acid oxidizing ability, and a test sample solution is applied to the two reaction reagent layers. Supplying and reacting the test sample solution with the enzyme in the presence of an electron acceptor, and electrochemically measuring the electron acceptor reduced with the enzymatic reaction, the test sample It is characterized in that glucose and lactic acid in the liquid are each determined.
【００１２】[0012]
【発明の実施の形態】本発明に用いるバイオセンサは、
少なくとも測定極と対極からなる電極系２組を絶縁性の
基板の同一面上、または異なる面上に形成し、それぞれ
の電極系上に少なくとも酸化還元酵素を含む反応試薬層
を形成したものである。一つの反応試薬層には、グルコ
ース酸化能を有する酵素、もう一つの反応試薬層には、
乳酸酸化能を有する酵素を含ませることによって、被検
試料液中のグルコースおよび乳酸が一つのバイオセンサ
で精度よく定量することができる。電子受容体には、フ
ェリシアン化イオン、ｐ−ベンゾキノン、フェナジンメ
トサルフェート、フェロセンなど水溶性で、酵素−電極
間の電子移動を媒介しうる化合物を任意に使用できる。DESCRIPTION OF THE PREFERRED EMBODIMENTS The biosensor used in the present invention is
At least two pairs of electrode systems including a measurement electrode and a counter electrode are formed on the same surface or different surfaces of an insulating substrate, and a reaction reagent layer containing at least an oxidoreductase is formed on each electrode system. . In one reaction reagent layer, an enzyme having glucose oxidizing ability, in another reaction reagent layer,
By including an enzyme having lactic acid oxidizing ability, glucose and lactic acid in the test sample solution can be accurately quantified by one biosensor. As the electron acceptor, any compound that is water-soluble and can mediate electron transfer between an enzyme and an electrode, such as ferricyanide ion, p-benzoquinone, phenazine methosulfate, and ferrocene, can be used.
【００１３】このようにして作製されたバイオセンサの
２つの反応試薬層に、グルコースおよび乳酸を含む被検
試料液を含浸させると、グルコース酸化能を有する酵素
を含むグルコース反応試薬層ではグルコースが、乳酸酸
化能を有する酵素を含む乳酸反応試薬層では乳酸がそれ
ぞれ反応し、この酵素反応に伴ってそれぞれの反応試薬
層に含まれる電子受容体が還元される。そして、グルコ
ース反応試薬層下の電極系および乳酸反応試薬層下の電
極系によって、それぞれの酵素反応に伴って還元された
電子受容体を酸化し、このとき得られる酸化電流値と、
測定装置に記憶させておいた濃度−電流値の検量線の基
づいて、被検試料液中のグルコースの濃度と乳酸の濃度
を算出する。このようにして、被検試料液中のグルコー
スおよび乳酸の定量ができるため、被検試料液の成分組
成を迅速に評価することが可能となる。When the two reaction reagent layers of the biosensor thus prepared are impregnated with a test sample solution containing glucose and lactic acid, glucose is contained in the glucose reaction reagent layer containing an enzyme having glucose oxidizing ability. Lactic acid reacts in the lactic acid reaction reagent layer containing an enzyme capable of lactic acid oxidation, and the electron acceptor contained in each reaction reagent layer is reduced along with the enzymatic reaction. Then, the electrode system under the glucose reaction reagent layer and the electrode system under the lactic acid reaction reagent layer oxidize the electron acceptor reduced with each enzymatic reaction, and an oxidation current value obtained at this time,
The concentration of glucose and the concentration of lactic acid in the test sample solution are calculated based on the concentration-current value calibration curve stored in the measuring device. In this manner, glucose and lactic acid in the test sample solution can be quantified, so that the component composition of the test sample solution can be quickly evaluated.
【００１４】被検試料液として好適なものには、日本酒
や醤油などのグルコースおよびＬ−乳酸を含む醸造食品
が挙げられる。醸造食品において、グルコースは発酵の
原料であり、甘味の成分である。またＬ−乳酸は、味に
影響する重要な成分である。このような醸造食品は、時
間の経過とともに成分の組成が変化するため、醸造食品
中の２つの成分を同時に迅速、簡易、および高精度に定
量することは、品質評価をおこなう上で重要である。ま
た、乳酸菌等の菌を培養する液体培地の状態の評価に本
発明を用いると、迅速、簡易および高精度に、培地中の
グルコースおよびＬ−乳酸の定量が可能となり、培養の
進行度合いを評価したり、栄養源の補給の必要性の有無
などを判断できたりして都合がよい。Suitable test liquids include brewed foods containing glucose and L-lactic acid such as sake and soy sauce. In brewed foods, glucose is a raw material for fermentation and a sweet component. L-lactic acid is an important component that affects taste. In such a brewed food, the composition of the components changes with the passage of time. Therefore, it is important to quickly, simply, and accurately quantify the two components in the brewed food for quality evaluation. . In addition, when the present invention is used to evaluate the state of a liquid medium for culturing bacteria such as lactic acid bacteria, the amount of glucose and L-lactic acid in the medium can be quickly, easily and accurately determined, and the degree of progress of the culture can be evaluated. It is convenient to be able to judge the necessity of supplementing nutrients.
【００１５】[0015]
【実施例】以下に、具体的な実施例を挙げて本発明をよ
り詳細に説明する。図１は、本発明による液体の評価法
の一実施例として用いたバイオセンサの反応試薬層を除
いた分解斜視図である。ポリエチレンテレフタレートか
らなる絶縁性の基板１の片面に、スクリーン印刷により
銀ペ−ストを印刷しリ−ド２、３、４、５を形成した。
次に、樹脂バインダーを含む導電性カーボンペーストを
用いて測定極６および７を形成した。測定極６はリード
２、測定極７はリード４とそれぞれ接触している。次
に、絶縁性ペーストを用いて絶縁層８を形成した。絶縁
層８は測定極６、７の露出部分の面積を一定とし、かつ
リ−ド部２、３、４、５を部分的に覆っている。そし
て、樹脂バインダーを含む導電性カーボンペーストをリ
ード３、５と接触するように印刷して対極９、１０を形
成した。The present invention will be described below in more detail with reference to specific examples. FIG. 1 is an exploded perspective view of a biosensor used as one embodiment of a liquid evaluation method according to the present invention, from which a reaction reagent layer is removed. Silver paste was printed by screen printing on one surface of an insulating substrate 1 made of polyethylene terephthalate to form leads 2, 3, 4, and 5.
Next, measurement electrodes 6 and 7 were formed using a conductive carbon paste containing a resin binder. The measuring electrode 6 is in contact with the lead 2, and the measuring electrode 7 is in contact with the lead 4. Next, the insulating layer 8 was formed using an insulating paste. The insulating layer 8 keeps the area of the exposed portions of the measurement electrodes 6 and 7 constant and partially covers the leads 2, 3, 4, and 5. Then, a conductive carbon paste containing a resin binder was printed so as to be in contact with the leads 3 and 5 to form the counter electrodes 9 and 10.
【００１６】このようにして電極系を形成した絶縁性基
板１に、スペーサー１３と、空気孔１７と１８を備えた
カバー１４とを、図１中一点鎖線で示すような位置関係
をもって接着し、バイオセンサを作製する。カバーおよ
びスペーサに高分子など透明な材料を用いると、反応試
薬層の状態や被検試料液の導入状況を外部から容易に確
認することが可能である。また、カバーを装着するとカ
バーとスペーサによって出来る空間部の毛細管現象によ
って、被検試料液は試料液供給口１５、１６に接触させ
るだけの簡易操作で容易にセンサ内に導入される。The spacer 13 and the cover 14 having the air holes 17 and 18 are adhered to the insulating substrate 1 on which the electrode system has been formed in such a manner as shown by a dashed line in FIG. Create a biosensor. When a transparent material such as a polymer is used for the cover and the spacer, the state of the reaction reagent layer and the state of introduction of the test sample liquid can be easily checked from the outside. Further, when the cover is mounted, the test sample liquid is easily introduced into the sensor by a simple operation of contacting the sample liquid supply ports 15 and 16 due to the capillary action in the space formed by the cover and the spacer.
【００１７】図２は、図１のバイオセンサのスペーサー
とカバーを除いた縦断面図である。図１と同様にして、
絶縁性基板１上に電極系を形成し、測定極６、対極９か
らなる電極系上に電子受容体としてフェリシアン化カリ
ウムを含み、酵素としてグルコースオキシダーゼを含む
溶液を滴下し、乾燥して、反応試薬層１１を形成してい
る。また、測定極７、対極１０からなる電極系上に電子
受容体としてフェリシアン化カリウムを含み、酵素とし
てラクテートオキシダーゼを含む溶液を滴下し、乾燥さ
せて反応試薬層１２を形成している。被検試料液のセン
サ内への供給を円滑にするために、レシチンの有機溶媒
溶液を試料液供給口１５、１６から反応試薬層にわたる
部位に展開し、乾燥させてレシチン層を形成してもよ
い。FIG. 2 is a longitudinal sectional view of the biosensor of FIG. 1 excluding a spacer and a cover. As in FIG.
An electrode system is formed on the insulating substrate 1, and a solution containing potassium ferricyanide as an electron acceptor and glucose oxidase as an enzyme is dropped on an electrode system composed of a measurement electrode 6 and a counter electrode 9, and dried to form a reaction reagent. The layer 11 is formed. Further, a solution containing potassium ferricyanide as an electron acceptor and lactate oxidase as an enzyme is dropped on an electrode system composed of the measurement electrode 7 and the counter electrode 10 and dried to form a reaction reagent layer 12. In order to smoothly supply the test sample liquid into the sensor, an organic solvent solution of lecithin is spread from the sample liquid supply ports 15 and 16 to a portion extending from the reaction reagent layer, and dried to form a lecithin layer. Good.
【００１８】図３は、本発明の液体の評価法の他の実施
例として用いたバイオセンサの反応試薬層を除いた分解
斜視図である。ポリエチレンテレフタレートからなる絶
縁性の基板１の表面に、スクリーン印刷により銀ペ−ス
トを印刷しリ−ド２、３を形成した。次に、樹脂バイン
ダーを含む導電性カーボンペーストを用いて測定極６を
形成した。測定極６はリード２と接触している。次に、
絶縁性ペーストを用いて絶縁層８を形成した。絶縁層８
は測定極６の露出部分の面積を一定とし、かつリ−ド部
２、３を部分的に覆っている。そして、樹脂バインダー
を含む導電性カーボンペーストをリード３と接触するよ
うに印刷して対極９を形成している。また、基板１の裏
面に、表面と同様にして、リード４、５と、測定極７、
絶縁層８、および対極１０を形成している。このように
して電極系を形成した絶縁性基板１に、空気孔１７また
は１８を備えたカバー１４、および２つのスペーサー１
３を、図３中一点鎖線で示すような位置関係をもって接
着し、バイオセンサを作製する。FIG. 3 is an exploded perspective view of a biosensor used as another embodiment of the liquid evaluation method of the present invention, from which the reaction reagent layer is removed. Silver paste was printed on the surface of the insulating substrate 1 made of polyethylene terephthalate by screen printing to form leads 2 and 3. Next, the measuring electrode 6 was formed using a conductive carbon paste containing a resin binder. The measuring electrode 6 is in contact with the lead 2. next,
The insulating layer 8 was formed using an insulating paste. Insulating layer 8
Has a constant area of the exposed portion of the measuring electrode 6 and partially covers the leads 2 and 3. Then, a counter electrode 9 is formed by printing a conductive carbon paste containing a resin binder so as to be in contact with the leads 3. Also, on the back surface of the substrate 1, similarly to the front surface, the leads 4, 5 and the measuring electrode 7,
An insulating layer 8 and a counter electrode 10 are formed. The cover 14 having the air holes 17 or 18 and the two spacers 1
3 are bonded in a positional relationship as shown by a dashed line in FIG. 3 to produce a biosensor.
【００１９】図４は、図３のバイオセンサの縦断面図で
ある。図３と同様にして、絶縁性基板１の表裏面上に電
極系を形成している。そして、図２と同様にして、測定
極６、対極９からなる電極系上にフェリシアン化カリウ
ムとグルコースオキシダーゼを含む反応試薬層１１を形
成し、測定極７、対極１０からなる電極系上にフェリシ
アン化カリウムとラクテートオキシダーゼを含む反応試
薬層１２を形成している。FIG. 4 is a longitudinal sectional view of the biosensor of FIG. An electrode system is formed on the front and back surfaces of the insulating substrate 1 in the same manner as in FIG. Then, similarly to FIG. 2, a reaction reagent layer 11 containing potassium ferricyanide and glucose oxidase is formed on an electrode system composed of the measurement electrode 6 and the counter electrode 9, and potassium ferricyanide is formed on an electrode system composed of the measurement electrode 7 and the counter electrode 10. And a reaction reagent layer 12 containing lactate oxidase.
【００２０】《実施例１》図２の構成のバイオセンサを
以下のようにして作製した。図１の測定極６、対極９か
らなる電極系上に、フェリシアン化カリウムとグルコー
スオキシダーゼを水に溶解させた混合水溶液を滴下し、
乾燥して、グルコース反応試薬層１１を形成した。ま
た、図１の測定極７、対極１０からなる電極系上にフェ
リシアン化カリウムとラクテートオキシダーゼを水に溶
解させた混合水溶液を滴下し、乾燥して、乳酸反応試薬
層１２を形成した。次に、この基板１にスペーサ１３お
よびカバー１４を図１中一点鎖線で示すような位置関係
をもって接着しバイオセンサを作製した。Example 1 A biosensor having the structure shown in FIG. 2 was manufactured as follows. A mixed aqueous solution obtained by dissolving potassium ferricyanide and glucose oxidase in water is dropped on an electrode system including the measurement electrode 6 and the counter electrode 9 in FIG.
After drying, a glucose reaction reagent layer 11 was formed. Further, a mixed aqueous solution in which potassium ferricyanide and lactate oxidase were dissolved in water was dropped on an electrode system composed of the measurement electrode 7 and the counter electrode 10 in FIG. 1 and dried to form a lactic acid reaction reagent layer 12. Next, a spacer 13 and a cover 14 were adhered to the substrate 1 in a positional relationship as indicated by a dashed line in FIG. 1 to produce a biosensor.
【００２１】上記のようにして作製したバイオセンサを
測定装置に装着すると、測定装置はスタンバイ状態にな
った。続いて試料供給孔１５、１６に、グルコースおよ
びＬ−乳酸を含む醸造食品である日本酒を被検試料液と
して接触させた。被検試料液は、毛細管現象で速やかに
応試薬層１１および１２に供給された。反応試薬層が溶
解し、反応試薬層１１上では、日本酒中のグルコースの
みが選択的に反応し、反応試薬層１２上では、日本酒中
のＬ−乳酸のみが選択的に反応する。このとき、反応試
薬層中に共存させておいた電子受容体フェリシアン化カ
リウムが、酵素反応と同時に還元されてフェロシアン化
カリウムに還元される。When the biosensor manufactured as described above was mounted on the measuring device, the measuring device was in a standby state. Subsequently, sake, which is a brewed food containing glucose and L-lactic acid, was brought into contact with the sample supply holes 15 and 16 as a test sample liquid. The test sample liquid was quickly supplied to the reagent layers 11 and 12 by capillary action. The reaction reagent layer dissolves, and only glucose in the sake selectively reacts on the reaction reagent layer 11, and only L-lactic acid in the sake selectively reacts on the reaction reagent layer 12. At this time, the electron acceptor potassium ferricyanide coexisting in the reaction reagent layer is reduced at the same time as the enzymatic reaction to be reduced to potassium ferrocyanide.
【００２２】試料を導入し、一定時間経過後、対極９を
基準にして測定極６にアノード方向へ＋０．５Ｖの定電
圧を印加し、同時に、対極１０を基準にして測定極７
に、同様にして定電圧を印加してフェロシアン化カリウ
ムを酸化した。そして、一定時間後に測定極６と対極９
との間に流れる酸化電流値と、測定極６と対極１０との
間に流れる酸化電流値をそれぞれ測定し、あらかじめ求
めておいた検量線に基づいて、日本酒中のグルコースお
よび乳酸の濃度を算出した。このようにして、日本酒中
の２つの成分が同時に精度よく定量でき、いつでも迅
速、簡易に日本酒の品質の評価ができた。After a predetermined time has passed after the sample was introduced, a constant voltage of +0.5 V was applied to the measurement electrode 6 in the anode direction with reference to the counter electrode 9, and at the same time, the measurement electrode 7 was determined with reference to the counter electrode 10.
Then, a constant voltage was similarly applied to oxidize potassium ferrocyanide. After a certain time, the measuring electrode 6 and the counter electrode 9
And the oxidation current flowing between the measurement electrode 6 and the counter electrode 10 are measured, and the glucose and lactic acid concentrations in the sake are calculated based on the calibration curve obtained in advance. did. In this way, two components in sake could be simultaneously quantified with high accuracy, and the quality of sake could be evaluated quickly and easily at any time.
【００２３】《実施例２》図４の構成のバイオセンサを
以下のようにして作製した。実施例１と同様にして、図
３の測定極６、対極９からなる電極系上にグルコース反
応試薬層１１を形成し、測定極７、対極１０からなる電
極系上に乳酸反応試薬層１２を形成した。そして、基板
１に、スペーサー１３およびカバー１４を図３中一点鎖
線で示すような位置関係をもって接着しバイオセンサを
作製した。試料供給孔１５、１６に、被検試料として乳
酸菌を培養している液体培地を接触させ、毛細管現象で
速やかにグルコース反応試薬層１１および乳酸反応試薬
層１２に供給した。Example 2 A biosensor having the configuration shown in FIG. 4 was manufactured as follows. In the same manner as in Example 1, a glucose reaction reagent layer 11 is formed on the electrode system including the measurement electrode 6 and the counter electrode 9 shown in FIG. 3, and a lactic acid reaction reagent layer 12 is formed on the electrode system including the measurement electrode 7 and the counter electrode 10. Formed. Then, the spacer 13 and the cover 14 were adhered to the substrate 1 in a positional relationship as shown by a dashed line in FIG. 3 to produce a biosensor. A liquid culture medium in which lactic acid bacteria were cultured as a test sample was brought into contact with the sample supply holes 15 and 16 and quickly supplied to the glucose reaction reagent layer 11 and the lactic acid reaction reagent layer 12 by capillary action.
【００２４】グルコース反応試薬層１１上では、培地中
の乳酸菌の栄養源であるグルコースのみが選択的に反応
する。乳酸反応試薬層１２上では、グルコースを摂取し
た乳酸菌の発酵によって生成したＬ−乳酸のみが選択的
に反応する。試料を導入し、一定時間後、対極９を基準
にして測定極６にアノード方向へ＋０．５Ｖの定電圧を
印加し、同時に対極１０を基準にして測定極７に同様に
して定電圧を印加した。そして、一定時間後に測定極６
と対極９との間に流れる酸化電流値と、測定極７と対極
１０との間に流れる酸化電流値をそれぞれ測定し、あら
かじめ求めておいた検量線に基づいて、培地中のグルコ
ースおよび乳酸の濃度を算出した。このように培地中の
グルコースおよびＬ−乳酸の濃度を測定することによっ
て、培養の進行度合いが評価でき、さらに養源の補給の
必要性の有無などを判断することができた。On the glucose reaction reagent layer 11, only glucose, which is a nutrient of lactic acid bacteria in the medium, reacts selectively. On the lactic acid reaction reagent layer 12, only L-lactic acid generated by fermentation of lactic acid bacteria that has taken glucose reacts selectively. A sample is introduced, and after a certain time, a constant voltage of +0.5 V is applied to the measuring electrode 6 in the anode direction with reference to the counter electrode 9, and at the same time, a constant voltage is similarly applied to the measuring electrode 7 with reference to the counter electrode 10. did. After a certain period of time, the measuring electrode 6
An oxidation current value flowing between the measurement electrode 7 and the counter electrode 10 and an oxidation current value flowing between the measurement electrode 7 and the counter electrode 10 were measured, and glucose and lactic acid in the medium were determined based on a calibration curve obtained in advance. The concentration was calculated. As described above, by measuring the concentrations of glucose and L-lactic acid in the medium, the progress of the culture could be evaluated, and the necessity of supplementation of nutrients could be determined.
【００２５】[0025]
【発明の効果】上記のように、本発明によると、日本酒
などの醸造食品や、菌を培養する培地など時間の経過と
共に成分組成が変化する液体を迅速かつ簡便に定量し
て、被検試料液の評価をすることがきる。As described above, according to the present invention, a liquid whose component composition changes over time, such as a brewed food such as sake, a culture medium for culturing bacteria, and the like, can be quickly and simply quantified to obtain a test sample. The liquid can be evaluated.
【図１】本発明による液体の評価法の一実施例として用
いたバイオセンサの反応試薬層を除いた分解斜視図であ
る。FIG. 1 is an exploded perspective view of a biosensor used as one embodiment of a liquid evaluation method according to the present invention, from which a reaction reagent layer is removed.
【図２】同バイオセンサの、スペーサーおよびカバーを
除いた縦断面図である。FIG. 2 is a longitudinal sectional view of the biosensor, excluding a spacer and a cover.
【図３】本発明の液体の評価法の他の実施例として用い
たバイオセンサの反応試薬層を除いた分解斜視図であ
る。FIG. 3 is an exploded perspective view of a biosensor used as another embodiment of the liquid evaluation method of the present invention, from which a reaction reagent layer is removed.
【図４】同バイオセンサの縦断面図である。FIG. 4 is a longitudinal sectional view of the biosensor.
【図５】従来のバイオセンサの平面図である。FIG. 5 is a plan view of a conventional biosensor.
【図６】同バイオセンサの縦断面図である。FIG. 6 is a longitudinal sectional view of the biosensor.
１ 絶縁性の基板 ２、３、４、５ リード部 ６、７、１９ 測定極 ８ 絶縁層 ９、１０、２０ 対極 １１ グルコース反応試薬層 １２ 乳酸反応試薬層 １３ スペーサ １４ カバー １５、１６ 試料液導入口 １７、１８ 空気孔 １９ 測定極 ２０ 対極 ２１ 参照極 ２２ 反応試薬層 Reference Signs List 1 Insulating substrate 2, 3, 4, 5 Lead 6, 7, 19 Measuring electrode 8 Insulating layer 9, 10, 20 Counter electrode 11 Glucose reactant layer 12 Lactate reactant layer 13 Spacer 14 Cover 15, 16 Sample liquid introduction Mouth 17, 18 Air hole 19 Measurement electrode 20 Counter electrode 21 Reference electrode 22 Reagent layer
Claims (2)
は異なる面上に形成された２組の電極系、および前記２
組の電極系上にそれぞれ形成された２つの反応試薬層か
らなり、前記反応試薬層の一方が少なくともグルコース
酸化能を有する酵素を含み、前記反応試薬層の他方が少
なくとも乳酸酸化能を有する酵素を含むバイオセンサを
用い、被検試料液を前記２つの反応試薬層に供給して前
記被検試料液と前記酵素を電子受容体の存在下で反応さ
せる工程、および前記酵素反応に伴って還元された電子
受容体を電気化学的に測定する工程を含み、被検試料液
中のグルコースおよび乳酸の濃度をそれぞれ定量するこ
とを特徴とする液体の評価法。An insulating substrate; two sets of electrode systems formed on the same surface or different surfaces of the substrate;
The reaction system comprises two reaction reagent layers formed on a set of electrode systems, one of the reaction reagent layers contains an enzyme having at least glucose oxidizing ability, and the other of the reaction reagent layers has an enzyme having at least lactate oxidizing ability. Using a biosensor comprising: supplying a test sample solution to the two reaction reagent layers to cause the test sample solution and the enzyme to react in the presence of an electron acceptor; and A method for evaluating a liquid, comprising a step of electrochemically measuring the electron acceptor, and quantifying the concentrations of glucose and lactic acid in a test sample solution.
とし、乳酸が生産される培地を請求項１記載の液体の評
価法を用いて評価する培地の評価法。2. A method for evaluating a medium, wherein glucose is used as a feed and a medium for producing lactic acid is evaluated using the liquid evaluation method according to claim 1 as the culture proceeds.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP10153141A JPH11344460A (en) | 1998-06-02 | 1998-06-02 | Method for evaluating liquid and culture medium |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP10153141A JPH11344460A (en) | 1998-06-02 | 1998-06-02 | Method for evaluating liquid and culture medium |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH11344460A true JPH11344460A (en) | 1999-12-14 |
Family
ID=15555920
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP10153141A Pending JPH11344460A (en) | 1998-06-02 | 1998-06-02 | Method for evaluating liquid and culture medium |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH11344460A (en) |
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1998
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