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Publication numberCN1210979 A
Publication typeApplication
Application numberCN 98116831
Publication date17 Mar 1999
Filing date28 Jul 1998
Priority date28 Jul 1997
Also published asCN1110700C, DE69800546D1, DE69800546T2, EP0894869A1, EP0894869B1, US6258254
Publication number98116831.0, CN 1210979 A, CN 1210979A, CN 98116831, CN-A-1210979, CN1210979 A, CN1210979A, CN98116831, CN98116831.0
Inventors宫本佳子, 池田信, 吉冈俊彦, 南海史郎
Applicant松下电器产业株式会社
Export CitationBiBTeX, EndNote, RefMan
External Links: SIPO, Espacenet
Biosensor
CN 1210979 A
Abstract  translated from Chinese
本发明揭示了能够抑制血液中与底物共存的红细胞的影响,以高精度对底物进行定量的生物传感器。 Disclosed is possible to suppress the blood erythrocytes with a substrate coexistence influence, on a substrate with high accuracy quantitative biosensor. 该生物传感器具备形成于电绝缘性基板上的包括工作电极和对应电极的电极系,以及至少包含氧化还原酶和电子接受体的反应层,前述电子接受体为钠盐。 The biosensor is formed on the electrode system includes a working electrode and a counter electrode comprising on an electrically insulating substrate, and containing at least an oxidoreductase and an electron acceptor in the reaction layer precursor, the electron acceptor is a sodium salt.
Claims(3)  translated from Chinese
1. 1. 一种生物传感器,其特征在于,具备形成于电绝缘性基板上的包括工作电极和对应电极的电极系,以及至少包含氧化还原酶和电子接受体的反应层,前述电子接受体为钠盐。 A biosensor comprising an electrode system formed on the working electrode and a counter electrode comprising on an electrically insulating substrate, and containing at least an oxidoreductase and an electron acceptor in the reaction layer precursor, the electron acceptor is a sodium salt.
2. 2. 如权利要求1所述的生物传感器,其中的钠盐为铁氰化钠。 The biosensor as claimed in claim 1, wherein the iron salt is sodium cyanide.
3. 3. 如权利要求1所述的生物传感器,前述反应层中包含亲水性高分子。 The biosensor as claimed in claim 1, said reaction layer comprises a hydrophilic polymer.
Description  translated from Chinese
生物传感器 Biosensor

本发明涉及对生物体试样中的特定成分进行定量的生物传感器。 The present invention relates to a biological sample of a specific component in quantitative biosensors.

以往,对于试样中的特性成分,作为不需要对试样溶液进行稀释和搅拌就能够简单地进行定量的方式,揭示了以下所述的生物传感器(日本专利公开公报平3-202764号)。 In the past, the characteristics of the component in the sample, as a sample solution does not require dilution and mixing can be easily performed in a quantitative manner, revealing the biosensor described below (Japanese Patent Publication No. Hei 3-202764).

生物传感器是利用网版印刷等方法在电绝缘基板上形成由工作电极和对应电极组成的电极系,在上述电极系上形成由亲水性高分子和氧化还原酶及电子接受体组成的酶反应层的传感器。 The biosensor is by screen printing and other methods of forming an electrode system by the working electrode and a counter electrode formed on an electrically insulating substrate is formed of a hydrophilic polymer and an oxidoreductase and an electron acceptor consisting of enzyme reaction on the electrode system sensor layer.

如果在上述制得的生物传感器的酶反应层上滴下含有底物的试样溶液,则酶反应层会溶解,底物与酶反应而被氧化,同时电子接受体被还原。 If a sample solution containing a substrate is dropped on the enzyme reaction layer of the biosensor obtained, the enzyme reaction layer is dissolved, the substrate with the enzyme reaction is oxidized, while the electron acceptor is reduced. 酶反应结束后,使该被还原的电子接受体电化学氧化,从所得的氧化电流值能够求出试样溶液中的底物浓度。 After completion of the enzyme reaction, the reduced electron acceptor is electrochemically oxidized, resulting from the oxidation current value can be determined concentration of the substrate in the sample solution.

该生物传感器通过使用各种不同的氧化还原酶可对各种底物进行定量。 The biosensor by using various oxidoreductase various substrates can be quantified.

作为生物传感器的一个例子,对葡萄糖传感器进行说明。 As an example of a biosensor for glucose sensor will be explained.

作为对葡萄糖进行电化学定量的方法,一般公知的是葡萄糖氧化酶(EC1.1.3.4)和氧电极及过氧化氢电极组合的方法(例如,铃木周一编《(生物传感器》讲谈社)。 As a method for quantitative electrochemical glucose, generally known is glucose oxidase (EC1.1.3.4) and the oxygen electrode and method for hydrogen peroxide electrode combinations (for example, Suzuki Monday series "(Biosensor" Kodansha) .

葡萄糖氧化酶以氧为电子接受体将作为底物的β-D-葡萄糖氧化为D-葡糖酸-δ-内酯。 Glucose oxidase with oxygen as an electron acceptor as β-D- glucose oxidase substrate is -δ- D- gluconic acid lactone. 在该反应进行的同时,氧被还原为过氧化氢。 The reaction is carried out at the same time, oxygen is reduced to hydrogen peroxide. 通过氧电极测定此时的氧消耗量或通过过氧化氢电极测定过氧化氢的生成量来对葡萄糖进行定量。 Determination of the amount of hydrogen generated through the oxidation of glucose quantified at this time is measured by the oxygen electrode through oxygen consumption or hydrogen peroxide electrode.

但是,上述方法中,溶解氧浓度对测定对象有很大影响,且在无氧条件下不能够进行测定。 However, the above method, the dissolved oxygen concentration has a great influence on the measurement object, and in the absence of oxygen can not be measured.

所以,开发了不以氧为电子接受体,而是以铁氰化合物、二茂铁衍生物、醌类衍生物等有机化合物或金属配位化合物为电子接受体的新型葡萄糖传感器。 Therefore, the development of oxygen is not the electron acceptor, but as ferricyanide compound, ferrocene derivatives, quinone derivatives and the like organic compound or metal complex as the electron acceptor novel glucose sensor.

该类型的生物传感器可使已知量的葡萄糖氧化酶和电子接受体以稳定状态附载于电极上,能够使电极系和反应层以近乎干燥的状态合为一体。 This type of biosensor can make a known amount of glucose oxidase and an electron acceptor in a stable state annexed to the electrodes, enabling the electrode system and the reaction layer with almost dry state into one.

由于这种生物传感器是一次性的(用完即弃型),只要在被插入测定器的传感器切片中导入检体试样,就能够很容易地对底物浓度进行测定,所以,近年来倍受瞩目,被有效地用作为各种医疗器械的诊断手段。 Because of this biosensor is disposable (disposable type), as long as the sample introduced into the sample measuring device is inserted into the sensor slices, it can be easily measured on substrate concentration, therefore, in recent years, times by the attention, it is effectively used as a diagnostic tool in a variety of medical devices.

如上所述,被包含在反应层中的电子接受体可使用铁氰化合物、二茂铁衍生物、醌类衍生物等有机化合物或金属配位化合物,特别是使用其钾盐。 As described above, are contained in the reaction layer, the electron acceptor iron cyanide compounds can be used, ferrocene derivatives, quinone derivatives and the like organic compounds or metal coordination compounds, particularly the use of its potassium salt.

但是,由于生理浓度以上的钾离子对红细胞状态维持有影响,会引起红细胞的破裂,以及对血液试样感应的应答性降低。 However, due to the physiological concentration of potassium ions more influential on RBC state is maintained, it will cause the breakdown of red blood cells, and induction of the response to the blood sample is reduced. 所以,即使血液中的底物浓度相同,也会存在血液试样中的红细胞量不同和传感器的应答性产生差异的情况。 Therefore, even if the substrate concentration in the blood is the same, the situation will be different amounts of red blood cells in the blood sample and the response of the sensor of the presence of a difference.

本发明的目的是提供能够抑制血液中与底物共存的红细胞的影响,以高精度对底物进行定量的生物传感器。 The object of the present invention is to provide the ability to suppress the influence of red blood cells in blood coexist with the substrate, the substrate with high precision quantitative biosensor.

本发明的具备形成于电绝缘性基板上的包括工作电极和对应电极的电极系和至少包含氧化还原酶和电子接受体的反应层的生物传感器中,前述电子接受体使用的是钠盐。 An electrode system including a working electrode and a counter electrode of the present invention have formed on an electrically insulating substrate and a biosensor comprising at least an oxidoreductase and an electron acceptor in the reaction layer, the electron acceptor used is a sodium salt.

本发明的较好实施状态中所用的前述钠盐为铁氰化钠。 Preferred embodiment of the present invention, in the state with the aforementioned iron sodium salt.

图1表示本发明一个实施例中的除去反应层的生物传感器的分解斜视图。 Figure 1 shows an exploded perspective view showing the removal of the reaction layer of the biosensor according to an embodiment of the present invention.

图2表示同一生物传感器主要部分的纵剖面图。 Figure 2 shows the same biosensor longitudinal sectional view of a main part.

钠盐溶解于试样溶液而生成的钠离子很少会引起血液中的红细胞破裂。 Sodium salt dissolved in a sample solution and sodium ions generated rarely cause red blood cells rupture. 所以,将钠盐作为电子接受体使用时,能够抑制血液中红细胞量的差对传感器的应答特性的影响。 Therefore, when the sodium salt as an electron accepting substance use, can suppress the influence of the difference in the amount of red blood cells in the blood of the response characteristics of the sensor.

上述钠盐是指铁氰化钠、β-萘醌-4-磺酸钠等。 Above it refers to iron sodium cyanide, β- naphthoquinone 4-sulfonate and the like.

本发明能够对包含在血液中的成分进行定量,所以,作为氧化还原酶能够使用葡萄糖氧化酶、葡萄糖脱氢酶、醇氧化酶、醇脱氢酶、胆固醇氧化酶、胆固醇脱氢酶、乳酸氧化酶、乳酸脱氢酶、抗坏血酸氧化酶和胆红素氧化酶等。 The present invention is capable of ingredients contained in the blood were quantified, therefore, can be used as the oxidoreductase, glucose oxidase, glucose dehydrogenase, alcohol oxidase, alcohol dehydrogenase, cholesterol oxidase, cholesterol dehydrogenase, lactate oxidase enzyme lactate dehydrogenase, ascorbate oxidase, bilirubin oxidase.

通过使用这些酶能够构成葡萄糖传感器、醇传感器、胆固醇传感器、乳酸传感器、抗坏血酸传感器和胆红素传感器等。 These enzymes can be constructed by using a glucose sensor, an alcohol sensor, a cholesterol sensor, a lactate sensor, a bilirubin sensor, etc. and ascorbic acid sensor.

此外,为了保护形成于基板上的电极系表面不受酶的影响,最好是用亲水性高分子覆盖电极系。 In addition, in order to protect the surface of the electrode system formed on the impact on the substrate from the enzyme, it is best to cover the electrode system with a hydrophilic polymer.

上述亲水性高分子可使用选自羧甲基纤维素、羟乙基纤维素、羟丙基纤维素、羧乙基纤维素、聚乙烯基吡咯烷酮、聚乙烯醇、明胶及其衍生物、丙烯酸或其盐的聚合物、甲基丙烯酸或其盐的聚合物、淀粉及其衍生物、马来酸酐或其盐的聚合物中的一种。 The hydrophilic polymer may be selected from carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, carboxyethyl cellulose, polyvinyl pyrrolidone, polyvinyl alcohol, gelatin and derivatives thereof, acrylic acid or salt thereof, a polymer of methacrylic acid or salts thereof, starch and derivatives thereof, maleic anhydride polymer or a salt thereof.

作为氧化电流值的测定方法可使用只有工作电极和对应电极的二电极方式,或再加上参比电极的三电极方式,三电极方式更能够进行正确的测定。 As a method for measuring the oxidation current value may be used only a working electrode and a counter electrode two-electrode mode, or the reference electrode, three-electrode mode, three-electrode system enables more accurate measurement plus.

以下,列举具体实施例对本发明进行更为详细的说明。 The following, cite specific embodiments of the present invention will be described in more detail.

图1表示本发明的除去反应层的生物传感器的分解斜视图。 Figure 1 shows an exploded perspective view of the removal of the reaction layer of the biosensor of the present invention.

在由聚对苯二甲酸乙二醇酯构成的电绝缘性基板1上,利用网版印刷法涂刷上银糊状物,形成导电板2和3。 On an electrically insulating substrate polyethylene terephthalate constituted 1, by screen printing method painted silver paste, a conductive plate 2 and 3. 然后,在基板1上印刷含有树脂粘合剂的导电性石墨糊状物,形成工作电极4。 Then, the substrate 1 printed conductive graphite paste containing a resin binder, forming the working electrode 4. 该工作电极4与导电板2相接触。 The working electrode 4 is in contact with the conductive plate 2. 接着,在基板1上印刷绝缘性糊状物,形成绝缘层6。 Next, on the insulating substrate 1 printed paste to form an insulating layer 6. 绝缘层6覆盖在工作电极4的外周部,这样就能够确保工作电极4露出部分的面积。 The insulating layer 6 covering the outer peripheral portion of the working electrode 4, so that it is possible to ensure that the work area of the exposed portion of the electrode 4. 然后,在基板1上印刷含有树脂粘合剂的导电性石墨糊状物,使其与导电板3接触,形成环状对应电极5。 Then, the substrate 1 printed conductive graphite paste containing a resin binder, so that contact with the conductive plate 3, corresponding to the annular electrode 5 is formed. 接着,在由工作电极和对应电极组成的电极系上或其近旁形成反应层。 Next, on the electrode system by the working electrode and a counter electrode composed of a reaction layer formed in the vicinity thereof.

具有反应层的绝缘性基板1和具备空孔11的封盖9及隔板10按照图1中点划线所示的位置连接,制得生物传感器。 An insulating substrate having a reaction layer 1 and cover with air holes 9 and 11 of the bulkhead 10 crossed the position shown in Figure 1 is connected midpoint prepared biosensors. 为了在基板和封盖间形成试样溶液的供给通道,在隔板10上设置了缝隙13。12为该试样溶液供给通道的开口部。 In order to cover between the substrate and the sample solution supply path is formed in the partition plate 10 is provided for opening the slit 13.12 the sample solution supply pathway.

图2表示本发明的一个实施例的除去隔板、封盖的生物传感器的主要部分的纵剖面图,如图1所示,在形成电极系的电绝缘性基板1上形成了亲水性高分子层7、包含酶和电子接受体的反应层(含酶层)8,以及卵磷脂层8a。 Figure 2 shows an embodiment of a separator to remove the present invention, the closure of the vertical sectional view of a main part of the biosensor shown in Figure 1, the electrode system formed on an electrically insulating substrate 1 is formed of a hydrophilic high molecular layer 7, containing an enzyme and an electron acceptor in the reaction layer body (enzyme-containing layer) 8 and the lecithin layer 8a.

实施例1在图1的基板1上的电极系上滴下羧甲基纤维素(以下略称为CMC)水溶液,使其在50℃的暖风干燥机中干燥10分钟,形成CMC层7。 EXAMPLE 1 Carboxymethylcellulose dropped on the electrode system on the base plate 1 in FIG. 1 (hereinafter referred to CMC) aqueous solution, dried in warm air dryer 50 ℃ for 10 minutes to form a CMC layer 7. 然后,将200单位葡萄糖氧化酶和40μmol铁氰化钠溶解于1ml水中,调制成混合水溶液。 Then, 200 units of glucose oxidase and sodium cyanide were dissolved in 1ml 40μmol iron water, to prepare a mixed aqueous solution. 将5μl该混合水溶液滴在CMC层7上,在50℃的暖风干燥机中干燥10分钟,形成包含氧化还原酶(葡萄糖氧化酶)和电子接受体(铁氰化钠)的反应层8。 The 5μl the mixed aqueous solution was dropped on the CMC layer 7 and dried for 10 minutes in a warm air dryer 50 ℃, the formation containing an oxidoreductase (glucose oxidase) and an electron acceptor (sodium iron) of the reaction layer 8.

然后,将卵磷脂的甲苯溶液滴在反应层8上,使其干燥,形成卵磷脂层8a后,按照图1中点划线所示的位置连接封盖和隔板,制得葡萄糖传感器。 Then, the toluene solution of lecithin was dropped on the reaction layer 8 and dried to form the lecithin layer 8a, shown in dotted line in Figure 1 connecting the position of closure and a separator, to prepare a glucose sensor.

所用的试样溶液是葡萄糖浓度为300mg/dl、红细胞容积比(血细胞比容值)分别为0%(血浆)、25%、38%和50%的血液。 The sample solution used was a glucose concentration of 300mg / dl, red blood cell volume ratio (hematocrit value) is 0%, respectively (plasma), 25%, 38% and 50% of the blood.

通过试样溶液供给通道12导入3μl血细胞比容值为0%的试样溶液后,试样溶液到达空孔11,溶解了电极系上的CMC层7、反应层8和卵磷脂8a。 0% of the value of the sample solution through the sample solution supply pathway 12 into 3μl hematocrit, the sample solution reaches the air vent 11 and dissolved the CMC layer 7 on the electrode system, the reaction layer 8 and the lecithin 8a. 导入试样25秒钟之后,以电极系的对应电极5为基准,在工作电极加上+0.5V的额定电压,测定5秒钟之后的电流值。 After the introduction of the sample for 25 seconds, corresponding electrode system 5 as a reference, the working electrode plus + 0.5V nominal voltage, the measured current value after 5 seconds.

同样,分别测定血细胞比容值为25%、38%和50%的试样溶液的电流值。 Similarly, hematocrit were measured value of 25%, the current value of the sample solution 38% and 50% of the.

所得的电流应答值与试样中的血细胞比容值无关,是一定的。 The resulting current response value is independent of the hematocrit value of blood cells in the sample, is certain.

比较例1与实施例1相同,在图1的电极系上形成CMC层7。 Comparative Example 1 is the same as in Example 1, CMC layer 7 is formed on the electrode system of FIG. 然后,将200单位葡萄糖氧化酶和40μmol铁氰化钾溶解于1ml水中,调制成混合水溶液。 Then, 200 units of glucose oxidase and potassium ferricyanide 40μmol dissolved in 1ml of water, to prepare a mixed aqueous solution. 将5μl该混合水溶液滴在CMC层7上,在50℃的暖风干燥机中干燥10分钟,形成包含氧化还原酶(葡萄糖氧化酶)和电子接受体(铁氰化钾)的反应层8。 The 5μl the mixed aqueous solution was dropped on the CMC layer 7 and dried for 10 minutes in a warm air dryer 50 ℃, the formation containing an oxidoreductase (glucose oxidase) and an electron acceptor (potassium ferricyanide) a reaction layer 8.

接着,与实施例1同样,形成卵磷脂8a之后,制得葡萄糖传感器,测定传感器的应答特性。 Next, after the same manner as in Example 1 to form a lecithin 8a, obtained glucose sensor, measuring the response characteristics of the sensor.

电流应答值随着血细胞比容值的增加而减少。 Current response value increases the hematocrit value is reduced. 其比例如表1所示。 Proportions shown in Table 1. 将血细胞比容值为0%时的电流应答值规定为100。 The current response value hematocrit value of 0% is defined as 100.

表1 Table 1

实施例2与实施例1同样,在图1的电极系上形成CMC层7。 Example 2 and Example 1, CMC layer 7 is formed on the electrode system of Figure 1. 然后,将400单位乳酸氧化酶和40μmol铁氰化钠溶解于1ml水中,调制成混合水溶液。 Then, 400 units of lactate oxidase and 40μmol of sodium cyanide were dissolved in 1ml water and iron, to prepare a mixed aqueous solution. 将5μl该混合水溶液滴在CMC层7上,在50℃的暖风干燥机中干燥10分钟,形成包含氧化还原酶(乳酸氧化酶)和电子接受体(铁氰化钠)的反应层8。 The 5μl the mixed aqueous solution was dropped on the CMC layer 7 and dried for 10 minutes in a warm air dryer 50 ℃, the formation containing an oxidoreductase (lactate oxidase) and an electron acceptor (sodium iron) of the reaction layer 8.

与实施例1同样,形成卵磷脂层8a后,制得乳酸传感器。 After the same manner as in Example 1 to form a lecithin layer 8a, obtained by lactic acid sensor.

所用的试样溶液是乳酸浓度为50mg/dl,血细胞比容值分别为0%(血浆)、25%、38%和50%的血液。 The sample solution used is a lactic acid concentration of 50mg / dl, hematocrit values were 0% (plasma) and 25%, 38% and 50% of the blood.

然后,与实施例同样对传感器的应答特性进行测定,所得的电流应答值与血细胞比容值无关,是一定的。 Then, in Example of the response characteristics of the sensor measured the resulting current response value is independent of the hematocrit value, it is certain.

比较例2与实施例1同样,在图1的电极系上形成CMC层7。 Comparative Example 2 and Example 1, CMC layer 7 is formed on the electrode system of FIG. 然后,将400单位乳酸氧化酶和40μmol铁氰化钾溶解于1ml水中,调制成混合水溶液。 Then, 400 units of lactate oxidase and potassium ferricyanide 40μmol dissolved in 1ml of water, to prepare a mixed aqueous solution. 将5μl该混合水溶液滴在CMC层7上,在50℃的暖风干燥机中干燥10分钟,形成包含氧化还原酶(乳酸氧化酶)和电子接受体(铁氰化钾)的反应层8。 The 5μl the mixed aqueous solution was dropped on the CMC layer 7 and dried for 10 minutes in a warm air dryer 50 ℃, the formation containing an oxidoreductase (lactate oxidase) and an electron acceptor (potassium ferricyanide) a reaction layer 8.

然后,与实施例1同样,形成卵磷脂层8a后,制得乳酸传感器。 Then, the same as in Example 1 to form a lecithin layer 8a, obtained by lactic acid sensor. 与实施例2同样,对传感器的应答特性进行测定。 Example 2. Similarly, the response characteristics of the sensor is measured.

其结果是,电流应答值随着血细胞比容值的增加而减少。 As a result, the current response value increases the hematocrit value is reduced. 其比例如表2所示。 Proportions shown in Table 2. 将血细胞比容值为0%时的电流应答值定为100。 The current response value hematocrit value of 0% is defined as 100.

表2 Table 2

如上所述,本发明提供了能够不受血液中与底物共存的血细胞成分的影响,以高精度对底物进行定量的生物传感器。 As described above, the present invention provides the effect of the blood can not coexist with the substrate of the blood cell components, on a substrate with high accuracy quantitative biosensor.

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CN104142360A *27 Jan 201412 Nov 2014全医电股份有限公司Biosensor test strip
CN104169716A *11 Mar 201326 Nov 2014株式会社村田制作所生物传感器
Classifications
International ClassificationC12Q1/00, G01N27/327
Cooperative ClassificationC12Q1/004
European ClassificationC12Q1/00B4
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