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CN1280673A - 改进型电化学生物感受器测试条 - Google Patents

改进型电化学生物感受器测试条 Download PDF

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CN1280673A
CN1280673A CN 98811850 CN98811850A CN1280673A CN 1280673 A CN1280673 A CN 1280673A CN 98811850 CN98811850 CN 98811850 CN 98811850 A CN98811850 A CN 98811850A CN 1280673 A CN1280673 A CN 1280673A
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improved
electrochemical
biosensor
test
strip
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CN 98811850
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CN1173180C (zh )
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威廉·F·克里斯莫
奈杰尔·A·瑟里奇
丹尼尔·R·麦克明
埃里克·R·迪博尔德
理查德·J·博登斯登耶
R·戴尔·德尔克
戴维·W·伯克
家雄·贾森·侯
罗伯特·基切尔·厄尔
布赖恩·A·希尔德
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罗奇诊断公司
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001Enzyme electrodes
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00Investigating or analysing materials by the use of electric, electro-chemical, or magnetic means
    • G01N27/26Investigating or analysing materials by the use of electric, electro-chemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28Electrolytic cell components
    • G01N27/30Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327Biochemical electrodes electrical and mechanical details of in vitro measurements
    • G01N27/3271Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
    • G01N27/3272Test elements therefor, i.e. disposable laminated substrates with electrodes, reagent and channels
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by the preceding groups
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay
    • G01N33/543Immunoassay; Biospecific binding assay with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366Apparatus specially adapted for solid-phase testing
    • G01N33/54373Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438Electrodes
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00Chemistry: molecular biology and microbiology
    • Y10S435/817Enzyme or microbe electrode

Abstract

一种生物感受器,包括一个第一绝缘底片1,绝缘底片1有一个第一表面22和一个第二表面23,还包括凹边2,切口3以及通孔4。

Description

改进型电化学生物感受器测试条

发明的技术领域本发明涉及一种生物感受器,以及其对在液体中分析物检测或测量方面的应用。

这种测试条一直特别应用于测量人血中的葡萄糖含量。糖尿病患者和健康护理专业人士一直在应用这种测试条,用以监视他们的血液中葡萄糖水平。如果打算把测试条用作染剂的光度检测用途时,测试条通常和光反射度测定仪一起应用,或者,如果打算把测试条用作电活性化合物的检测用途时,测试条通常和测量某些电学性能,例如电流的测定仪一起应用。

然而,先前制作的测试条对使用它们的人们来说存在某些问题。例如,测试条相对比较小而视力受损的糖尿病患者对于把血样正确地加到测试条的样品施用区是有很大困难的。所以,将测试条制成便于视力受损的人能够方便地加量到测试条,则会是很有用的。

当测试条是毛细状充填器时,也就是,当测试条的化学反应腔是在毛细状空间时,随着待试液样均匀而充分地注入腔内可能会出现一些特别的问题。由于毛细状空间很小同时制作测试条所用的材料成分,使试样也许会暂停进入毛细状反应腔。另外,还可能将不足的试样汲入毛细状反应腔,因此得到不准确的试验结果。假如能够将上述问题减少到最低限度则会是非常有用的。

最后,各种测试条,特别是为糖尿病患者测量血内葡萄糖含量所用的那些测试条都是大批量生产的。用于制作这些测试条的工艺过程,例如机械冲压,能导致干涸在测试区的表面上的测试试剂裂化或破碎,由此导致测试条内的试剂耗损或试剂的不正确放置。因而,设计一种能够经得住例如机械冲压那样的加工工序的测试试剂也会是很有用的。

本发明的电化学生物感受器测试条提供了现有技术中所发现的上述各种问题的解决办法。

发明的概括说明本发明是一种改进的、具有四种新颖的、有很大优点特征的电化学生物感受器测试条。

第一新颖特征是为视力受损的人们或用于低或无采光情况下,供易于识别试样施用口用的,沿测试条边缘的一个凹边。

测试条有一毛细状测试腔,而所述测试腔的顶片包括生物感受器测试条的第二新颖特征。第二新颖特征是起到“充填至此”线作用的透明或半透明窗口,以此识别已将足够的试样(一种液体试样,例如血液)加入到测试腔内,以便准确完成测试。上述窗口指定准确完成一项测试所需要的最低限度的试样量或加入量,因此,提供可见的准确性,它降低由于测试条加量不足而造成错误测试结果的概率。

上述窗口的长度和宽度比毛细状测试腔的长度和宽度稍短些。窗口被定尺寸并设置成使其遮盖工作电极的整体宽度,并至少遮盖计数器或生物感受器测试条的基准电极宽度的10%左右。最好,将窗口周围的顶片区按如下方式着色,即当通过窗口观察时,试样与窗口周围的顶片区之间提供良好的颜色对比度以便于识别测试条的充分加量。

测试条的第三新颖特征是包含位于试样施用口处的一个切口,或多个切口。在测试条的第一绝缘底片和顶面两者内都生成切口。这些切口被定尺寸并设置成使得它们在测试条中一个遮盖另一个。这些切口可减少一种叫做“加量暂停”的现象。当将试样加到无切口测试条的试样施用口时,试样能暂停地进入毛细状测试腔。这种“加量暂停”增加测试时间。当测试条包含切口时,就降低了加量暂停。另外,在第一绝缘底片和顶面两者内都包含切口时,使其能够从各种不同的角度使试样到达试样施用口。假如仅在顶面内有切口,则使试样到达的角度会受到限制。

最后,测试条的第四新颖特征是试剂,它包括从约100千道尔顿到约900千道尔顿平均分子量、浓度从约0.2%(重量∶重量)到2%(重量∶重量)的聚乙烯氧化物,它使干燥的试剂更有亲水性和坚实。在包含聚乙烯氧化物的情况下,测试试剂能够更容易地耐受住在测试条组装时的机械冲压和由测试条用户机械性拨弄。另外,包括从约1.75%(重量∶重量)到17.5%(重量∶重量)的聚乙烯氧化物的干燥的试剂,当将含水的试样加到测试条的测试腔时,能够容易地再溶解,或再悬浮。

附图的简短说明图1是本发明的一较佳实施例的部件分解图。

图2显示一完全组装好的较佳测试条。

图3a-3i表示一种制作本发明测试条的较佳方法。

图4是图2的测试条通过剖线28-28的剖面图。

图5是图2的测试条通过剖线29-29的剖面图。

图6说明不同组类的测试条假设的校准曲线。

如图1所示,将导电轨条5和6布置在第一绝缘底片1的第一表面22上。轨条5可以是一工作电极,用像钯,铂,金,碳和钛这样一类的导电材料制成。轨条6可以是一计数器电极,用像钯,铂,金,银,含银合金,镍铬合金,碳,钛和铜这样一类的导电材料制成。首选贵金属是因为它们提供更为稳定、具有再生性的电极表面。特别首选钯,因为它是更加难于氧化的贵金属之一,还由于它是一种相对廉价的贵金属。

最好,将导电轨条5和6熔敷在例如聚酰亚胺或聚酯的绝缘底板上,以便在测试条的加工和制作时降低开裂电极材料的可能性。上述导电轨条的一个实例是在UPILEX聚酰亚胺底板上具有表面电阻小于每平方(persquare)5欧姆的钯镀层,它可从美国加利福尼亚Courtalds-AndusPerformance Films in Canoga Park购得。

导电轨条5和6表示生物感受器测试条的电极。这些电极必须充分间隔开,以便在一个电极的电化学过程与在另一个电极的电化学过程不相干扰。电极5和6之间的较佳距离大约为1.2毫米(mm)。

在图1示出的测试条中,导电轨条5应当是工作电极,而导电轨条6应当是计数器电极或基准电极。轨条6假如由典型的基准电极材料制成,例如银/氯化银,应当是基准电极。在最佳实施例中,轨条5是由钯制成的工作电极,而轨条6也是由钯制成的计数器电极而且实际上与工作电极的尺寸相同。

三电极布置也是可能的,其中测试条包括一外加的安置在导电轨条6与通孔4之间的导电轨条。在三电极布置中,导电轨条5应当是工作电极,轨条6应当是计数器电极,而在导电轨条6与通孔4之间的第三电极则应当是基准电极。

复盖导电轨条5和6的是第二绝缘基片7,第二绝缘基片7是由与第一绝缘底片1类似的或最好是相同的材料制成。基片7有第一表面8和第二表面9。用一种粘合剂,例如一种热熔胶将第二表面9固定到底片1的表面和导电轨条5和6上。上述胶的一个实例是DYNAPOL S-1358胶,可从Hüls美国,Inc,220 Davidson Street,PO Box 6821,Somerset,NJ08873购得。基片7还包含第一开口10和第二开口11。第一开口10的导电轨条5和6的露出部分与测定仪电连接,该测定仪在用测试条的试样试剂混合以后测定试样的某些电性能。第二开口11露出导电轨条5和6的不同部分,用于把测试试剂12馈给到轨条5和6的那些露出的表面上。(在图1中,由开口11露出导电轨条5和6的整个宽度。然而,也可能仅露出导电轨条6的部分宽度,该导电轨条6或是计数器电极或是基准电极,只要至少由开口11露出其宽度的大约10%.)另一方面,第二绝缘基片7包含在图1中示出的与凹边2重合的凹边19。

测试试剂12是供由测试条加以完成测试用的特定的试剂。可以将试剂12加到通过第二开口11所指定的区域中,导电轨条5和6的整个露出的表面区上。在该区中,试剂12的其它馈给方式也是可能的。例如,倘若在测试条的该区中导电轨条6有一像银/氯化银这样的基准电极结构,那么,测试试剂12可以只需要遮盖在该区中导电轨条5的露出区域。另外,可以不需要用测试试剂复盖住电极的整个露出区域,只要用试剂遮盖往电极的很好指定和具有再生性的区域就行。

遮盖第一表面8的一部分和第二开口11的是顶片13。顶片13包含凹边14和切口15。将凹边14和切口15加工并设置成使得它们直接遮盖住凹边2和19,以及切口3。顶片13可以用塑性材料制成,例如用从约2密耳到6密耳厚度的透明或半透明聚酯薄片制成。顶片13有第一表面16和第二表面17。将顶片13的第二表面17用一种适当的粘合剂,例如3 M 9458丙烯酸粘合剂,它可从3M,Identification andConverter Systems Division,3M Center,Building 220-7W-03,St.Paul,MN 55144购得。

最好,顶片13还包含透明或半透明的窗口18。将窗口18定尺寸并设置成使得当顶片被固定到第二基片7上时,该窗口遮盖住导电轨条5的整个宽度和至少导电轨条6宽度的10%左右。

顶片13的第二表面17,开口11的边缘和绝缘基片7的第一表面22(以及固定到底片1的第一表面22上的导电轨条5和6)限定了一毛细状测试腔。上述毛细状腔的长度和宽度是由开口11的长度和宽度限定的,而上述腔的高度则是由第二绝缘基片7的厚度限定的。

较佳的测试条可以如同图3a-3i说明的工艺过程所示那样加以制作。用热熔胶(DYNAPOL S-1358,可从Hüls购得)涂在一张绝缘片材21(MELINEX 329,7密耳厚,可从ICI购得)的侧面上。(图3a)沿着线24切割片材21,由此形成在第一表面22上涂有胶的第一绝缘底片1和在第二表面段9上涂有胶的第二绝缘基片7。(图3b和3c)用模压在基片7内冲口形成第一开口10和第二开口11。(图3d)下一步,将用钯在Upilex底板(可从Courtalds-Andus Performance Films购得)上制成的导电轨条5和6从卷筒上转下,预先切割成大约为1.5毫米宽并平铺在底片1的表面22上,使得所述Upilex底板靠近表面22。将基片7的表面9靠近底片1的表面22和导电轨条5和6处放置,由此形成图3e中示出的夹层结构。上述夹层结构是经过热密封处理的。

然后,将测试试剂12配合到开口11内并使之干燥。(图3f)在试剂经过干燥后,用模压冲孔建立通孔4。(图3g)下一步,将包含亲水性镀层25和窗口18的顶片13按如下方式放在开口11的上面,即使得窗口18遮盖住导电轨条5的整个宽度并遮盖住导电轨条6宽度的一半左右。松开衬垫脱开顶片13并如图3h所示将其粘合固定到表面8上。

最后,如图3i中所示,用冲模冲压出专用的测试条。上述冲模可以冲压出带或不带切口15的测试条。如果包含切口15时,则顶角的较佳角度为105°。其它角度,例如从大约45°到大约105°对切口来说也都是可能的。另外,切口15可以是单个切口或多个切口。

如上所述,将测试试剂12配合到由开口11所指定的测试条的区域内。在上述制作工艺过程中,在放入测试试剂12之前最好对开口11作电晕放电处理。应用电晕放电处理用来增大开口11所露出的部分表面22及导电轨条5和6的表面能量,促使试剂12的均匀分布,同时预先清洁由开口11所露出的部分导电轨条5和6。已经发现对导电轨条5和6预先清洁会显著地改进测试条的性能。可以用电弧间隙大约为1毫米(0.040英寸),按从大约每厘米每秒20到90瓦特(W/CM/S)的功率密度范围来施加电晕放电处理。

在首选方法中,以裹覆方式,在上述功率密度下,如图3e中示出的表面之上作电晕放电处理。如果对试剂12施加在5分钟之内进行所述处理是最有效的,一般,对试剂12施加在45秒钟之内实施所述处理。

为了确保试剂12充分地接合在开口11中,并且对表面8不会比开口11所露出的表面22及导电轨条5和6部分有更大的亲合力,在表面8上降低电晕放电处理的效应是有利的。结合一种能选择性地降低以裹覆方式电晕放电处理过程效应的电晕放电消除处理,以降低在开口11壁板外部(测试条被处理层面)的面积上处理的效应。上述电晕放电消除处理包括供应去离子水的薄膜,使得水接触表面8,但将不接触开口10和11。可借助“油心”效应墨滚,曲面印刷,或其它市场上可获得的涂敷方法来完成水薄膜的应用,它最好是从大约1.5微米到大约3.0微米厚度(大约每平方米9.1克水)。然后,应用强制热对流或红外方法正好在试剂12应用之前从表面干燥所述水薄膜。上述处理的基本效应在于有效地将表面8的表面能量在试剂12应用之前,降低到小于62达因,而在开口11内区域的表面仍保持在电晕放电处理后的表面能量。

在该较佳实施例中,按测量人血试样中葡萄糖含量选定测试试剂12的配方。以下即将示出利用酶奎诺蛋白质(包含吡咯并喹啉醌(PQQ))葡糖脱氢酶和氧化还原介体铁氰化物制备一升最佳葡糖试剂的议定书。(奎诺蛋白质葡糖脱氢酶是Enzyme Commission No.1.1.99.17.)步骤1:在去离子水中准备一种NATROSOL溶液。这是通过把0.45克(g)NATROSOL-250M(从Aqualon可购得的一种微晶羟乙基纤维素)加到414g去离子水中,同时在不少于30分钟的一个周期内,以每分钟不小于250转(rpm)的速度搅动而完成的。最好用一个悬吊的旋转搅拌叶轮使用三或四叶片的涡轮型螺旋桨来完成混合。上述螺旋桨尺寸和外形的选定主要取决于所用混合容器的半径。选定的螺旋桨的半径一般大于混合容器半径的75%。

步骤2:把5.6g AVICEL RC-591F(可从FMC Corp.购得的一种微晶纤维素)按照步骤1所述的溶液中,在不少于60分钟、以不小于570rpm的速度进行混合的同时,用分散渐加方式将上述AVICEL加进溶液中。

步骤3:把8.4g聚乙烯氧化物(300千道尔顿平均分子量)在不少于45分钟的一个周期内、以不小于690rpm的速度进行混合的同时,逐渐加进按照步骤2所述的混合物中。

步骤4:通过把12.1g磷酸氢一钾(无水的)和21.3g磷酸氢二钾(无水的)加到450g去离子水中配制出一种缓冲溶液。

步骤5:从步骤4的配制中取出50g缓冲溶液试样。把12.5mg辅酶PQQ(可从Fluka购得)加到上述50g试样中。搅拌上述溶液直到辅酶完全溶解。(制备酶,最好是用磁性搅拌棒和磁性搅拌板)。

步骤6:把1.21兆单元奎诺蛋白质葡糖脱氢酶的脱辅基酶在以低速(在磁性搅拌板上小于400rpm)进行搅拌以防止发泡的同时,逐渐加进按照步骤5所述的溶液中。在不少于2小时的情况下对上述合成溶液加以混合,以便使酶和辅酶的缔合趋于稳定,由此合成一种奎诺蛋白质葡糖脱氢酶溶液。

步骤7:把59.1g铁氰化钾加到按照步骤4所述的缓冲溶液中。然后再加入6.2g琥珀酸钠。混合上述溶液直到全部溶质完全溶解为止。在溶解以后,鉴定溶液的PH值并要求其大致为6.76(误差为±0.05)。

步骤8:把按照步骤7所述的溶液在以不小于190rpm的速率混合的同时,逐渐加入按照步骤3所述的混合物中。

步骤9:把8.20g海藻糖在不少于10分钟的一个周期内、以不大于190rpm的速率进行混合的同时加到按照步骤8所述的混合物中步骤10:把0.35g TRITON X-100表面活性剂(可从BoehringerMannheim Biochemicals购得)在不大于190rpm的速率进行混合的同时,加到按照步骤9所述的混合物中。上述混合物必须在不少于5分钟内连续进行混合。

步骤11:把按照步骤6所述的酶溶液加到按照步骤10所述的混合物中,并在不少于30分钟的一个周期内、以不小于190rpm的速率混合现今已完全的试剂。

步骤12:制作所需要的设备,对上述试剂加以过滤,使其通过100微米筛袋或通过100微米滤过器汇集到泵唧系统。

以上已详细说明的奎诺蛋白质葡糖脱氢酶的脱辅基酶,可从德国的Boehringer Mannheim GmbH(Boehringer Mannheim GmbH商标标号1464221)获得。另一方面,可以通过Duine等人列举的以下议定书FEBSLETTERS,vol.108,no.2,pps.443-46从Acinetobacter Calcoaceticus获得上述脱辅基酶。

使Acinetobacter Calcoaceticus生长在用0.02摩尔(M)琥珀酸钠或0.10M乙醇、处于22℃具有良好通气条件下增补的岩盐介质上。在对数生长期的末期收获晶胞,并能获得约4g/l的湿晶胞收率。

将冻结晶胞(10g)融化并与15毫升(ml)36毫摩尔(mM)Tris/39mM甘氨酸缓缓混合。在加入6毫克(mg)溶菌酶以后,对悬浮物在室温下搅拌15分钟、并在48,000Xg条件下作离心处理10分钟。用包含1%TRITON X-100表面活性剂的36毫摩尔(mM)Tris/39mM甘氨酸缓缓排除上层清液并两次抽提丸片。使上层清液的各离心步骤综合并直接使用。

把胞外提取物加到二乙氨乙基葡聚糖凝胶柱(DEAE-Sephacel column)(13×2.2厘米(cm)),用包含1%TRITON X-100表面活性剂的36毫摩尔(mM)Tris/39mM甘氨酸缓冲加以补偿,并用同一缓冲去清洗柱。上述酶并不附着于柱材料上,用2M乙酸将化合活性部分滴定到pH 6.0。把上述溶液直接加到CM-琼脂糖凝胶的柱上CL-6B(5×1cm),并用5mM磷酸钾(pH 6.0)加以平衡。在用同一缓冲去清洗柱直到在洗出液中不存在TRITON X-100表面活性剂以后,用0.1M磷酸钾(pH 7.0)将酶洗脱。

然后,在72小时4℃条件下,倚着包含3M溴化钾的0.1M乙酸钠(pH 4.5)渗出酶。接着,用12小时倚着0.02M磷酸钾(pH 7.0)渗析出酶,结果形成脱辅基酶。

在较佳测试条中,开口11约为3.2毫米×6.7毫米左右。在葡萄糖测试条的最佳实施例中,把借助以上议定书制成的4.5毫升测试试剂加到开口11中。(参见图3f)上述试剂数量将充分复盖住导电轨条5和6的露出表面。然后,测试试剂12在大约70℃ 1到2分钟左右、得到干燥。

上述形成的较佳干燥的葡萄糖试剂薄膜每克试剂将包含从大约2,000到9,000酶的活性单元。较佳的试剂每克将包含如下另外一些组分:62.2毫克(mg)聚乙烯氧化物3.3mg NATROSOL 250 M41.5mg AVICEL RC-591 F89.4mg磷酸氢一钾157.9mg磷酸氢二钾437.3mg铁氰化钾46.0mg琥珀酸钠148.0mg海藻糖2.6mg TRITON X-100表面活性剂。

重要的是,包括按重量从大约0.2%到按重量大约2%的聚乙烯氧化物,其平均分子量为从大约100千道尔顿到大约900千道尔顿,最好按重量大约为0.71%的聚乙烯氧化物,其平均分子量为300千道尔顿,在以上所指的湿试剂中,提供一种测试试剂,在干燥时,对用机械冲压那样的加工方法的测试条来说是坚实的,对测试条用户的机械性拨弄来说也是坚实的,而且,当把例如人血那样的液态试样加到其上时会再溶解或再悬浮。在干燥以后,聚乙烯氧化物的百分比范围从大约1.75%(重量∶重量)到大约17.5%(重量∶重量)。在较佳的干燥试剂中,聚乙烯氧化物的百分比大约为6.2%(重量∶重量)。

较佳干燥的葡萄糖试剂的厚度将是这样,使其与测试化学的固有性能相组合,以调节测试的灵敏度,防止血球比容计偏差的干扰。在本发明的上述最佳实施例中,薄膜厚度(取湿试剂分配容积与由开口11所露出表面区域之比的形式)是使得将4.5毫升试剂分配到大致22.5平方毫米的面积(较佳开口11的面积)内。具有上述厚度的薄膜中,包含聚乙烯氧化物其平均分子量为从大约100千道尔顿到大约900千道尔顿,当在测定人血试样中的葡萄糖时,结果得到拥有降低的对血球比容计偏差灵敏度的感受器。

测试试剂12在开口11中干燥以后,将顶片13放在开口11的上面,并如上所述用粘合剂将其固定到表面8上。顶片13本身是按照以下描述的生产过程单独加工的。

最好,顶片13是由MELINEX 561聚酯薄膜制成的,其厚度为5mil。在型板27中的第一表面16上大范围地印刷不透明的油墨,使得窗口18保持透明或半透明。将窗口18设置并定尺寸为,当顶片13被固定到表面8上时,使其如图3h所示与开口11对准。

在第二表面17上,用叠层的粘合物系统在于可以使顶片最大地固定到表面8上。上述粘合物系统能够方便地是例如可从许多商业来源购得丙烯酸粘合剂,但最好是来自3M Inc.部件号为9458的产品。

此外,在把顶片放在表面8上之前,铺一层透明或半透明的塑料片(最好是一种聚乙烯对苯二酸酯(PET),例如从大约0,001到大约0.004英寸厚的Melinex S塑料)对着第二表面17上的粘合物系统放置,使其对准并延伸到窗口18的范围那边。上述复盖的塑料是亲水性的覆盖片25。按特性选择覆盖片25以便把亲水特征给与毛细状测试腔的内表面,从而促使例如血之类的液体试样流进测试腔内。覆盖片25能够从许多可购得的呈现亲水表面的覆盖片中加以选择,但最好是产品号为ARCARE 8586可从Adhesives Research,Inc.购得的产品。覆盖片25还起到阻止顶片的粘合剂与试剂12直接接触的作用。

最后,将顶片13放到表面8上。(参见图3h)在此阶段,顶片13上没有印上油墨的透明或半透明窗口18必须如图3h所示与开口11对准。应当这样来选定透明或半透明窗口18的尺寸,使在下面的毛细状通道(大于大约75%)宽度的实际部分通过窗口可以看得到。窗口18的矩形尺寸应当露出工作电极5的整个宽度。因此,当例如血的试样被加入毛细状测试腔时,通过试样施用口20,对于有适当视力敏锐度的用户来说就有可能确定所述窗口是否完全充满试样。通过如刚才说明的那样选择窗口尺寸,就有可能为测试条的用户提供测试条已充分加满试样的反馈。窗口装满正被视见证实从而提供保证试样已遮盖住工作电极的主要面积,同时也遮盖住计数器或基准电极6的主要部分。就毛细状填料电化学生物感受器而言,由试样形成电极的遮盖率对取得精确的测试是很重要的。上述测试条的充分加量的视见证实相对由于未检测到测试条加量不足而造成的错误测试结果提供一种安全保障。

完成的测试条26在把试样加到试样施用口20以后,与能够测定测试条某些电性能的测定仪结合应用。(参见图2)可以测定电性能,例如,电流,电位,电荷,或阻抗。美国专利No.5,413,690说明测定电位变化以完成一种分析测试的一个实例,该专利所公开的内容本文结合作为参考文献。

美国专利Nos.5,288,636和5,508,171说明测定电流以完成一种分析测试的一个实例,这些专利所公开的内容本文结合作为参考文献。

在最佳实施例中,把测试条26连接到测定仪,它包括电源(蓄电池)。在美国专利Nos.4,999,632;5,243,516;5,366,609;5,352,351;5,405,511;和5,438,271中可发现在这类测定仪和生物感受器方面的各种改进,这些专利所公开的内容本文结合作为参考文献。

可以用本发明的电化学测试条分析许多包含液体的分析物。例如,可以测定人体液中的分析物,诸如全血,血清,尿和脑脊髓液中的分析物。同样,也可测定在发酵产品中,以及可能包含在环境污染的环境物质中发现的分析物。

为了用如上所述的较佳测试条确定在人血试样中的葡萄糖浓度,其中轨条5和6是基本上相同尺寸的钯,而葡萄糖试剂是以上所说明的试剂,可把血样加到试样施用口20。上述试样借助毛细状作用将被吸入测试腔内。一旦进入测试腔内,血样将与测试试剂12混合。经过一定的时间例如30秒钟的诱导期以后,将由测定仪的电源把电位差加在轨条5和6之间。在最佳实施例中,所加的电位差是300毫伏。在加上300毫伏的电位差以后可在从0.5秒到大约30秒的任何时间内测定电流。可以使所测定的电流与血样中的葡萄糖浓度相关连。

在从液体试样鉴定分析物期间,可以通过电流测定仪借助算法的应用使所测定的电流与试样中分析物的浓度相关连。上述算法如下实例所示可以是一项简单的算法:[分析物]=Ci7.5+d式中[分析物]代表试样中分析物的浓度(参见图6),i7.5是在电极之间加上电位差应用以后7.5秒钟时测定的电流(以毫安为单位),C是线条30(图6)的斜率,而d则是轴线相交(图6)。

通过用已知的分析物浓度作出测定,可形成校准曲线30(图6)。上述校准将被存储在测定仪的只读存储器(ROM)键中,并将可应用于测试条的特定组类。图6中线条31和32代表用于测试条的两种其它不同组类的另外假设的校准曲线。用于这些生物感受器组类的校准对以上算法中的C和d产生略微不同的值。

在用于从人们全血的试样中作葡萄糖分析的较佳方法中,在电极之间加上电位差以后,从3秒到9秒每隔0.5秒作出电流测定。使这些电流测定与血样中葡萄糖的浓度相关连。

在上述从血样中测定葡萄糖的实例中,按不同的时间作出电流测定(在电位差施加以后,从3秒到9秒),而不是按单一固定的时间(如上文所描述的),于是合成的算法也更为复杂,可用如下的方程式加以表示:[葡萄糖]=C1i1+C2i2+C3i3+…Cnin+d,式中i1是在第一测量时间(在应用300毫伏电位差以后3秒钟)测定的电流,i2是在第二测量时间(在应用300毫伏电位差以后3.5秒钟)测定的电流,i3是在第三测量时间(在应用300毫伏电位差以后4秒钟)测定的电流,in是在第n测量时间(在该实例中,在应用300毫伏电位差以后第13测量时间或9秒钟)测定的电流,C1,C2,C3,以及Cn是从例如主要组分分析或部分最小二乘方多变量回归分析方法导出的系数,而d则是回归相交(按葡萄糖浓度单位)。

另一种方法是,可以借助图形显示电流对一定时间间隔(例如在应用300毫伏电位差以后从3秒到9秒)的测量时间所生成的曲线进行积分,由此获得在测量段期间传送的总电荷来确定被测试样中葡萄糖的浓度。上述传送的总电荷与被测试样中葡萄糖的浓度直接成正比。

另外,还可以对实际测定时环境温度与完成校准时环境温度间的差异加以校正。例如,倘若在环境温度为23℃的情况下形成用于葡萄糖测定的校准曲线的话,则可应用以下方程式来校正葡萄糖测定:[葡萄糖]校正=[葡萄糖]测定X(1-K(T-23℃)),式中T是在试样测定时的环境温度(按℃为单位),而K则是从以下回归方程中导出的常数:Y=K(T-23),式中为了要计算出K值,由测定仪在各种不同的温度T情况下,以及在23℃(基准情况)时测定大量的葡萄糖浓度。然后,根据T-23完成Y的线性回归。K值是上述回归的斜率。

可以把本发明的各种各样的特征结合到其它电化学测试条中,例如,在美国专利Nos.5,120,420;5,141,868;5,437,999;5,192,415;5.264,103;以及5,575,895公开的那些测试条中,这些专利所公开的内容本文结合作为参考文献。

Claims (32)

1.一种测试条,其特征在于,它包含:一第一绝缘底片,有第一和第二表面和沿一边缘的凹边及一通孔;至少两个导电轨条,固定到第一绝缘底片的第一表面上;一第二绝缘基片,有第一和第二表面、一个类似于第一绝缘底片凹边的凹边及第一和第二开口,上述第二表面被固定到导电轨条和第一绝缘底片的第一表面上并被定位成使第二绝缘基片的凹边遮盖住第一绝缘底片的凹边,第一开口露出的部分导电轨条用于与能够测定电性能的测定仪电连接,第二开口露出导电轨条的不同部分和通孔;测试试剂,至少遮盖住由第二开口露出的部分导电轨条;以及一顶片,有第一和第二表面及一个类似于第一和第二绝缘基片凹边的凹边,顶盖的第二表面被固定到第二绝缘基片的第一表面上并设置成使得1)顶片的第二表面和第一绝缘底片的表面形成一毛细状充填腔的相对两壁,2)顶片的凹边遮盖住第一和第二绝缘基片的凹边。
2.一种测试条,其特征在于,它包含:一第一绝缘底片,有第一和第二表面和沿一边缘的切口及一通孔;至少两个导电轨条,固定到第一绝缘底片的第一表面上;一第二绝缘基片,有第一和第二表面及第一和第二开口,上述第二表面被固定到导电轨条和第一绝缘底片的第一表面上,第一开口露出的部分导电轨条用于与能够测定电性能的测定仪电连接,第二开口露出导电轨条的不同部分、在第一绝缘底片中的切口和通孔;测试试剂,至少遮盖住由第二开口所露出的部分导电轨条;以及一顶片,有第一和第二表面及沿边缘的切口,顶片的第二表面被固定到第二绝缘基片的第一表面上并被设置成使得1)顶片的第二表面和第一绝缘底片的第一表面形成毛细状充填腔的相对两壁,以及2)顶片的切口遮盖住在第一绝缘底片中的切口,由此,当液态试样接触到在第二绝缘基片中的第二开口处测试条的边缘时,在顶片中的切口和在第一绝缘底片中的切口将促使其流入毛细状腔内而没有显著的暂停现象。
3.一种测试条,其特征在于,它包含:一第一绝缘底片,有第一和第二表面及通孔;至少两个导电轨条,固定到第一绝缘底片的第一表面上;一第二绝缘基片,有第一和第二表面及第一和第二开口,上述第二表面被固定到导电轨条和第一绝缘底片的第一表面上,第一开口露出的部分导电轨条用于与能够测定电性能的测定仪电连接,第二开口露出第一和第二导电轨条的不同部分和通孔;测试试剂,至少遮盖住由第二开口露出的部分第一和第二导电轨条;以及一顶片,有第一和第二表面及一个透明或半透明窗口,顶片的第二表面被固定到第二绝缘基片的第一表面上并被设置成使得顶片的第二表面和第一绝缘底片的第一表面形成毛细状充填腔的相对两壁,并使透明或半透明窗口定尺寸并设置成,使得窗口从把试样加入到测试条处的边缘延伸,并遮盖住导电轨条之一的整个宽度和另一导电轨条的至少10%宽度左右。
4.一种供测试条用的试剂,其特征在于,它包含:适用于完成一项测试的反应组分,以及包括按重量从大约0.2%到按重量大约2%、其平均分子量为从大约100千道尔顿到大约900千道尔顿的聚乙烯氧化物的并可形成薄膜的可溶解或可悬浮的混合物,其中可按湿态把试剂加到测试条内,接着使其干燥,然后当把含水试样加到干燥的试剂上时使之再溶解或再悬浮。
5.按照权利要求1所述的测试条,其特征在于,它另外包含:沿第一绝缘底片中凹边的第一切口和沿顶片中凹边的一个切口,第一和第二两个切口都被设置成使它们相互遮盖。
6.按照权利要求1所述的测试条,其特征在于,顶片有透明或半透明的窗口,该窗口被定尺寸和设置成,使得窗口遮盖最紧靠第一绝缘底片凹边的导电轨条的整个宽度和另一导电轨条宽度的至少10%。
7.按照权利要求5所述的测试条,其特征在于,顶片有透明或半透明的窗口,该窗口被定尺寸和设置成,使得窗口遮盖最紧靠第一绝缘底片凹边的导电轨条的整个宽度和另一导电轨条宽度的至少10%。
8.按照权利要求1所述的测试条,其特征在于,该测试试剂包括适用于完成一项测试的反应组分,以及包括按重量从大约0.2%到按重量大约2%、其平均分子量为从大约100千道尔顿到大约900千道尔顿的聚乙烯氧化物的并可形成薄膜的可溶解或可悬浮的混合物,其中可按湿态把试剂加到测试条内,接着使其干燥,然后当把含水试样加到干燥的试剂上时使之再溶解或再悬浮。
9.按照权利要求5所述的测试条,其特征在于,该测试试剂包括适用于完成一项测试的反应组分,以及包括按重量从大约0.2%到按重量大约2%、其平均分子量为从大约100千道尔顿到大约900千道尔顿的聚乙烯氧化物的并可形成薄膜的可溶解或可悬浮的混合物,其中可按湿态把试剂加到测试条内,接着使其干燥,然后当把含水试样加到干燥的试剂上时使之再溶解或再悬浮。
10.按照权利要求6所述的测试条,其特征在于,该测试试剂包括适用于完成一项测试的反应组分,以及包括按重量从大约0.2%到按重量大约2%、其平均分子量为从大约100千道尔顿到大约900千道尔顿的聚乙烯氧化物的并可形成薄膜的可溶解或可悬浮的混合物,其中可按湿态把试剂加到测试条内,接着使其干燥,然后当把含水试样加到干燥的试剂上时使之再溶解或再悬浮。
11.按照权利要求7所述的测试条,其特征在于,该测试试剂包括适用于完成一项测试的反应组分,以及包括按重量从大约0.2%到按重量大约2%、其平均分子量为从大约100千道尔顿到大约900千道尔顿的聚乙烯氧化物的并可形成薄膜的可溶解或可悬浮的混合物,其中可按湿态把试剂加到测试条内,接着使其干燥,然后当把含水试样加到干燥的试剂上时使之再溶解或再悬浮。
12.按照权利要求1所述的测试条,其特征在于,所述顶片的第二表面包括一个亲水性的覆盖片。
13.按照权利要求5所述的测试条,其特征在于,所述顶片的第二表面包括一个亲水性的覆盖片。
14.按照权利要求6所述的测试条,其特征在于,所述顶片的第二表面包括一个亲水性的覆盖片。
15.按照权利要求7所述的测试条,其特征在于,所述顶片的第二表面包括一个亲水性的覆盖片。
16.按照权利要求8所述的测试条,其特征在于,所述顶片的第二表面包括一个亲水性的覆盖片。
17.按照权利要求9所述的测试条,其特征在于,所述顶片的第二表面包括一个亲水性的覆盖片。
18.按照权利要求10所述的测试条,其特征在于,所述顶片的第二表面包括一个亲水性的覆盖片。
19.按照权利要求11所述的测试条,其特征在于,所述顶片的第二表面包括一个亲水性的覆盖片。
20.按照权利要求7所述的测试条,其特征在于,该测试试剂包括适用于测试的反应组分,以及包括按重量从大约0.2%到按重量大约2%、其平均分子量为300千道尔顿的聚乙烯氧化物的并可形成薄膜的可溶解或可悬浮的混合物。
21.按照权利要求20所述的测试条,其特征在于,该聚乙烯氧化物按重量大约为0.71%。
22.一种供测试条用的试剂,其特征在于,它包含:适用于完成一项测试的反应组分,以及按重量从大约1.75%到按重量大约17.5%、其平均分子量为从大约100千道尔顿到大约900千道尔顿的聚乙烯氧化物,其中当把含水试样加到试剂上时,试剂将再溶解或再悬浮。
23.按照权利要求1所述的测试条,其特征在于,该测试试剂包括适用于完成一项测试的反应组分,以及按重量从大约1.75%到按重量大约17.5%、其平均分子量为从大约100千道尔顿到大约900千道尔顿的聚乙烯氧化物,其中当把含水试样加到试剂上时,试剂将再溶解或再悬浮。
24.按照权利要求5所述的测试条,其特征在于,该测试试剂包括适用于完成一项测试的反应组分,以及按重量从大约1.75%到按重量大约17.5%、其平均分子量为从大约100千道尔顿到大约900千道尔顿的聚乙烯氧化物,其中当把含水试样加到试剂上时,试剂将再溶解或再悬浮。
25.按照权利要求6所述的测试条,其特征在于,该测试试剂包括适用于完成一项测试的反应组分,以及按重量从大约1.75%到按重量大约17.5%、其平均分子量为从大约100千道尔顿到大约900千道尔顿的聚乙烯氧化物,其中当把含水试样加到试剂上时,试剂将再溶解或再悬浮。
26.按照权利要求7所述的测试条,其特征在于,该测试试剂包括适用于完成一项测试的反应组分,以及按重量从大约1.75%到按重量大约17.5%、其平均分子量为从大约100千道尔顿到大约900千道尔顿的聚乙烯氧化物,其中当把含水试样加到试剂上时,试剂将再溶解或再悬浮。
27.按照权利要求26所述的测试条,其特征在于,聚乙烯氧化物的平均分子量为300千道尔顿。
28.按照权利要求27所述的测试条,其特征在于,在试剂中聚乙烯氧化物的数量按重量大约为6.2%。
29.一种借助电晕放电处理有选择地增大表面亲水性的方法,其特征在于,它包含:把功率密度大约从每厘米每秒20到90瓦特的电晕放电电弧施加到表面上;接着,有选择性地把水膜加到需要消除电晕放电处理效应的区域上;以及然后通过干燥去除上述水份。
30.按照权利要求29所述的方法,其特征在于,施加大约从1.5微米到大约3.0微米厚度的水膜。
31.按照权利要求30所述的方法,其特征在于,所述水是去离子水。
32.按照权利要求31所述的方法,其特征在于,所述电晕放电电弧是在距离表面大约0.040英寸处施加的。
CN 98811850 1997-12-05 1998-12-02 改进型电化学生物感受器测试条 CN1173180C (zh)

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