CN1214117C - 基于四氮唑化合物的诊断 - Google Patents

基于四氮唑化合物的诊断 Download PDF

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CN1214117C
CN1214117C CNB011162511A CN01116251A CN1214117C CN 1214117 C CN1214117 C CN 1214117C CN B011162511 A CNB011162511 A CN B011162511A CN 01116251 A CN01116251 A CN 01116251A CN 1214117 C CN1214117 C CN 1214117C
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T·欧阳
Y·S·于
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Abstract

适合于测量含血红蛋白的生物流体例如全血中的分析物浓度的试剂。该试剂含有对分析物有特异性的依赖黄素的酶，黄素辅因子，条件是且仅是黄素不和酶结合，四氮唑染料前体，电子转移剂和亚硝酸盐。该试剂形成染料，据此可测定分析物浓度。亚硝酸盐抑制由血红蛋白非酶造成的对染料形成的干扰。优选，试剂用于干条中以测量全血中的葡萄糖。

Description

基于四氮唑化合物的诊断
本发明是1999年3月30日申请的共同待审的美国申请序号09/282083的继续申请，该共同待审的申请是美国申请序号为09/161876(申请日1998年9月28日)的继续申请，是现在的美国专利US5902731。
本发明涉及允许在含血红蛋白的生物流体中测量分析物浓度的诊断组合物。该组合物基于四氮唑染料前体并涉及抑制血红蛋白诱导的它们的还原。
脂肪组织是人体中能量贮存最丰富的形式之一。它释放贮存的脂肪酸进入主要由肝代谢的循环系统。在该过程中，脂肪被消耗，能量被释放，被人体加以利用。通常，消耗很少的脂肪，脂肪酸完全代谢为二氧化碳和水，该转化不会打乱身体的敏感的pH平衡。然而，如果身体中的碳水化合物的量不充足，例如由于饮食，那么脂肪消耗和脂肪酸的产生可增加至潜在危害的水平。除了饮食外，依赖胰岛素的病人由于它们的碳水化合物的代谢不足而易受到伤害。当使用过量的脂肪酸以提供身体的能量需求时，会产生大量的乙酰乙酸盐、丙酮和β-羟丁酸盐。这些中间产物称为酮体，疾病是已知的酮酸中毒。
酮体通常由身体再循环为其它形式，只要它不是令人无法忍受的。因此，健康的人积聚了可忽略量的这些分析物。当大量的脂肪在相对短的时间被代谢或当大部分的能量来自脂肪时，产生大量的酮体。如果不立刻校正的话，这些脂肪代谢物的过量产生导致一定的神经性紊乱。
酮体存在于血中，并且如果超过阈值，则通过尿排泄。通过先进的临床分析仪器可容易地检测它们。β-羟丁酸盐、乙酰乙酸盐和丙酮的百分率分别平均为78％、20％和2％。由于其相对低的浓度和高挥发性，所以丙酮很少被检测到。但是，通过硝普盐反应可定量确定乙酰乙酸盐，用酶法定量β-羟丁酸盐。利用乙酰乙酸盐试验条已经数十年。它们基于硝普盐离子和醛以及酮的偶合反应。让碱性尿样或血清样品和硝普盐反应几分钟，形成紫色。颜色的强度表明乙酰乙酸盐浓度。但是，丙酮干扰该试验，导致较高的读数。另外，当病人从酮酸中毒恢复时，尿中和血中的乙酰乙酸盐含量增加，因此使诊断困难。
β-羟丁酸盐试验对于控制酮体浓度更有用。它基于β-羟丁酸盐和相应的脱氢酶在烟酰胺腺嘌呤二核苷酸(NAD)辅因子存在下的氧化反应。(严格地说，仅D-β-羟丁酸盐天然存在和被氧化，但为了简洁，在整个说明书和所附的权利要求书中，我们省略了“D”。)氧化时，产生NADH，用UV分光光度计直接测量其浓度。因此，在光谱中相应的信号改变和分析物的浓度成比例。不幸的是，在UV区域中发生NADH激发；因此该检测方式仅适合于实验室仪器。另一个控制β-羟丁酸盐的方法是用四氮唑化合物氧化NADH。
四氮唑化合物一般对强碱和光很敏感。因此，需特别注意保证这些化合物的完整性。然而，四氮唑化合物在研究组织代谢中起着重要的作用。例如，该类化合物已用于探测细胞中不需氧氧化和还原反应。另外，它们一般用于临床诊断。该类化合物典型地是在还原剂存在下进行还原反应以得到高度着色的甲的浅颜色或无色化合物。还原剂例如抗坏血酸盐、巯基化合物或NADH、NADPH、PQQH2(还原的PQQ-吡咯并喹啉醌)、FMNH2(还原的FMN-黄素单核苷酸)和FADH2(还原的FAD-黄素腺嘌呤二核苷酸)的变体能够形成染料。
在临床诊断中，已发现这些染料在厌氧反应中对监测由它们的母化合物NAD(P)+形成NAD(P)H是非常有价值的。(参见，例如US5360595，1994年11月1日授予D.Bell等人。)氧化还原反应很快，对氧不敏感。得到的染料颜色很强烈，具有低的水溶性。
一般来说，可使用四氮唑染料前体测量全血中的酮体和葡萄糖。但是，如果血红蛋白不含在血的红细胞中，则可通过血红蛋白(Fe(II))非酶还原四氮唑形成有色的甲。因此，游离的血红蛋白会严重干扰检测。事实上，由于溶血和产生相对于目的分析物大量的游离血红蛋白。因此，在典型的酮体测量中，来自血红蛋白的干扰信号会超过目的信号。相反，葡萄糖的测量，特别是在正常浓度或超过正常浓度时，则不受影响。当在高血细胞比容样品中或在较高温度下进行该反应时，血红蛋白氧化反应较快，同样严重干扰葡萄糖的测量。既然不易避免造成游离血红蛋白存在的红细胞的溶血，所以，如果用四氮唑分析，则必须在试验前从样品中除去红细胞。
通过用膜和过滤器过滤、化学试剂捕集或这两种方法的结合，从样品中除去红细胞。从全血中分离红细胞的过滤方法昂贵，需要相当大的样品容积。使用过滤以从全血样品除去红细胞的血酮(β-羟丁酸盐)试验的实例是从GDSDiagnostics，Elkhart，IN得到的KetoSite试验。(参见由C.Burtis等人编辑的TietzTextbook of Clinical Chemistry，2nd Ed.，W.B.Saunders Co.，Philadelphia，PA，1994，第974页)用在那个试验中的“试验卡”具有两个过滤层，使得卡相当昂贵和需要大量的(25μL)的血样品。另外，该血必须没有发生溶血。
过滤和化学捕集的结合用于得自Miles的AmesGlucometer EncoreTM血糖条中。该条利用过滤材料层和凝集助剂(土豆凝集素)以从红细胞中除去干扰。(参见Chu等人的欧洲专利申请0638805A2，1995年2月15日公开)
系统中引进氧化剂以氧化血红蛋白形成高铁血红蛋白是降低血红蛋白干扰的另一种方式。尽管已知高铁氰化物将血红蛋白转化为高铁血红蛋白，但它们也破坏所需产品即NADH。
Palmer等人的EPO0330517B2(公开于1989年8月30日)公开了一种测量生物化学分析物的方法，包括分析物和能够具有电子转移酶活性的氧化酶反应以产生还原的酶。比色法分析该酶以确定分析物浓度。该酶反应是不依赖氧的。
Freitag等人的WO94/01544(公开于1994年1月20日)公开了一种用于分析物分析的稳定的试剂。该试剂包括酶、吩嗪衍生物、四氮唑盐和二价金属盐以稳定该试剂。
Storhoff等人的WO94/01578(公开于1994年1月20日)也公开了一种用于分析物分析的稳定的试剂。该试剂包括酶、介质、四氮唑盐和稳定该试剂的氧化剂。
本发明提供一种在含血红蛋白的生物流体中测量分析物浓度的试剂。该试剂包括：
a)具有与之结合的黄素和对分析物具有特异性的依赖黄素的酶，
b)四氮唑染料前体，
c)电子转移剂，和
d)亚硝酸盐。
在本发明的另一个实施方案中，该试剂包括：
a)对分析物具有特异性的和没有与之相结合的黄素的依赖黄素的酶，
b)黄素单核苷酸(FMN)或黄素腺嘌呤二核苷酸(FAD)，
c)四氮唑染料前体，
d)电子转移剂，和
e)亚硝酸盐。
该试剂特别适合于在一个或多个底物上涂敷以形成干的试剂条，用于测量含血红蛋白的生物流体中的分析物浓度。特别优选的试剂条包括：
a)载体层，
b)在载体层上的具有涂层的垫片，包括
i)具有与之结合的黄素和对分析物具有特异性的依赖黄素的酶，
ii)四氮唑染料前体，和
iii)电子转移剂，和
c)在试验垫片上的用亚硝酸盐涂敷的吸湿顶层。
本发明的另一种条包括
a)载体层，
b)在载体层上的具有涂层的垫片，包括
i)对分析物具有特异性的和没有与之相结合的黄素的依赖黄素的酶，
ii)FMN或FAD，
iii)四氮唑染料前体，
iv)电子转移剂，和
c)在试验垫片上的用亚硝酸盐涂敷的吸湿顶层。
图1是本发明的试验条的透视图。
图2是本发明另一种试验条的分解图。
图3是本发明再一种试验条的分解图。
图4是本发明的葡萄糖分析的化学图示。
图5是显示亚硝酸盐作为血红蛋白抑制剂在双层分析上的效果图。
图6是显示亚硝酸盐作为血红蛋白抑制剂在单层葡萄糖分析上的效果图。
本发明通过产生还原形式的辅因子如NADH、NAD(P)H、PQQH2、FMNH2或FADH2，提供了一种测量含血红蛋白的生物流体(如全血)中的分析物浓度的试剂，所述辅因子是分析物浓度的量度。试剂中包含的亚硝酸盐克服了血红蛋白对测量还原辅因子浓度的干扰。对测量但不限于酮体和葡萄糖特别有用。
图1描述了本发明的典型试验条10，它由附着在载体14上的试验垫片12组成。载体是塑料的，例如聚苯乙烯、尼龙或聚酯或金属片或任何其它本领域公知的适合材料。用试剂涂敷该试验垫片，该试剂和分析物反应产生颜色变化。该试验垫片优选包括吸湿材料，例如滤纸或聚合物膜。但是，由于反应不需要氧气，所以该试验垫片可以是非吸湿材料，例如塑料膜。试剂包括对分析物有特异性的酶，氢阴离子转移剂，四氮唑染料前体，适当的酶辅因子和血红蛋白抑制剂。为了较高的稳定性，任选还包括缓冲剂和稳定剂。
如图2所示，试验条也可以是多层结构，上层16覆盖在试验垫片12上。在该结构中，试剂被分为2层。例如，血红蛋白抑制剂可以涂敷在任选的上层16上，剩余的试剂涂敷在试验垫片12上。优选上层16是吸湿性的，并用作扩散层和吸附层以吸附过量的样品。样品被涂敷在上层16，它通过而达到试验垫片12。通过测量整个载体层14上的颜色改变，或如果层14邻接反应区处是不透明的，通过任选的窗口或通过孔18，确定分析物浓度。
在图3所示的另一个实施方案中，间隔20分开了上层16和试验垫片12。间隔20优选是非吸湿的塑料膜，在其两面具有黏附层(未示出)。间隔20中的通道22给样品提供了毛细路径以从开口24流至测量区26。流动取决于试验垫片12的表面和邻接层之间的排气口，或者选择性的孔18。通过选择性的孔/窗口18监测测量区域26中的颜色改变。试剂可以都在试验垫片12上，或在试验垫片和非吸湿层14和16之一或两者之间分配。因此，试剂的第一部分在试验垫片上，试剂的第二部分在非吸湿层之一上或两层都有。当我们称试剂为“涂层”和“在”层上时，我们意在包括试剂将被吸附进层内的可能性，特别是如果该层是吸湿性的。
所有依赖黄素的酶适合于本发明的分析。适合的氧化酶和它们相应的分析物包括：醇的醇氧化酶，葡萄糖的葡萄糖氧化酶，半乳糖的半乳糖氧化酶，胆固醇的胆固醇氧化酶，L-乳酸盐的L-乳酸盐氧化酶，尿酸的尿酸盐氧化酶，胆红素的胆红素氧化酶，胆碱的胆碱氧化酶。适合的脱氢酶和相应的分析物包括：丙酮酸盐的丙酮酸脱氢酶，D-乳酸的D-乳酸脱氢酶和琥珀酸的琥珀酸脱氢酶。
当不和酶结合时，必须添加辅因子以活化该酶。可以添加到依赖黄素的酶中的辅因子包括：黄素单核苷酸(FMN)和黄素腺嘌呤二核苷酸(FAD)。在酶的存在下，分析物还原了辅因子。
染料形成方法中的下一步是通过电子转移剂从还原的辅因子吸取氢阴离子(hydride)。适合的电子转移剂包括黄递酶例如硫辛酸脱氢酶、铁氧化还原蛋白-NADP-还原酶和脂酰胺脱氢酶。当使用黄素辅因子时，更优选是非酶电子转移剂，例如吩嗪硫酸甲酯(PMS)、吩嗪硫酸乙酯(PES)、1-甲氧基吩嗪硫酸甲酯或Meldola Blue。反应动力学和稳定性是选择电子转移剂或“氢阴离子吸取剂(hydride abstractor)”的主要因素。例如PMS是通用的氢阴离子吸取剂，因为它和下列的大多数四氮唑化合物具有相对快的反应动力学。因此，当辅因子是PQQ时，优选PMS。但是PMS比酶基的氢阴离子吸取剂对光更敏感。为此，当辅因子是NAD时，黄递酶更稳定，且是优选的。
被捕获的氢阴离子转移至四氮唑化合物(染料前体)，形成有色的甲。最适合该装置的四氮唑化合物是：2-(2′-苯并噻唑基)-5-苯乙烯基-3-(4′-邻苯二甲酰肼基)四氮唑(BSPT)、2-苯并噻唑基-(2)-3，5-二苯基四氮唑(BTDP)、2，3-二(4-硝基苯基)四氮唑(DNP)、2，5-二苯基-3-(4-苯乙烯基苯基)四氮唑(DPSP)、二苯乙烯基氮蓝四唑(DSNBT)、3，3′-[3，3′-二甲氧基-(1，1′-联苯基)-4，4′-二基]-双[2-(4-硝基苯基)-5-苯基(-2H)四氮唑(NBT)、3-(4，5-二甲基-2-噻唑基)-2，5-二苯基-2H四氮唑(MTT)、2-苯基-3-(4-羧苯基)-5-甲基四氮唑(PCPM)、四氮唑蓝(TB)、硫代氨基甲酰氮蓝四唑(TCNBT)、四氮蓝四唑(TNBT)、四氮唑紫(TV)、2-苯并噻唑噻唑基-3-(4-羧基-2-甲氧基苯基)-5-[4-(2-磺基乙基氨基甲酰基)苯基]-2H-四氮唑(WST-4)和2，2′-二苯并噻唑基-5，5′-双[4-二(2-磺基乙基)氨基甲酰基苯基]-3，3′-(3，3′-二甲氧基-4，4′-亚联苯基)二四氮唑二钠盐(WST-5)。优选使用水溶性染料前体，更优选WST-5，以便和生物样品相容。另外，当使用WST-5时，得到的甲化合物在紫-蓝区域显示出强的光谱吸收，因此降低了对来自血红蛋白的背景信号的校正的需求。
最后，血红蛋白抑制剂存在于试剂中以消减血红蛋白和四氮唑化合物之间的不希望的染料形成反应。血红蛋白抑制剂的作用是氧化血红蛋白为不与四氮唑或甲反应的高铁血红蛋白。令人惊奇地是，亚硝酸盐，例如亚硝酸钠、亚硝酸钾和它们的衍生物在抑制血红蛋白方面是非常有效的，而不会破坏还原的辅因子(例如NADH、PQQH2、FMNH2或FADH2)。亚硝酸盐在高温和血细胞比容样品下同样是有效的。优选亚硝酸钠，因为它具有高的水溶性，无毒和相对便宜。
选择性地，该试剂还包括稳定剂，例如二价金属盐。
尽管本发明的试剂以湿的化学模式使用，例如在优选的实施方案中在比色杯中，但本发明提供了在全血中分析β-羟丁酸盐或葡萄糖的干条。该条可以是单层的或两层的。两层条由膜试验垫片组成，该垫片优选是尼龙制成的，放在载体和上层之间。载体优选是由聚酯构成。上层可以是网眼的或任何本领域公知的吸收材料。优选的材料是用甲基油酰基牛磺酸钠处理的多孔聚乙烯(购自Fairburn，GA的Porex公司)。我们称该材料为“Porex”。优选该试验垫片具有正电荷表面。更优选，该试验垫片是聚酰胺。试验垫片含有包含葡萄糖氧化酶(包括黄素辅因子)、PMS(或其同系物的一种)和WST-5的试剂(下面的表1)。Porex上层含有亚硝酸盐试剂(表2)。
在单层条中，省略了Porex层，包括亚硝酸盐的整个试剂(表3)涂在试验垫片上。注意到在两层条和单层条中，可以使用黄素与之结合的依赖黄素的酶或没有黄素与之结合的依赖黄素的酶。在后一情况下，添加黄素辅因子(例如FMN或FAD)。
在操作中，使用者将一滴全血涂在Porex上层的上表面。当全血或溶解的血和Porex接触时，亚硝酸钠被复制并和可利用的游离血红蛋白反应，因此使得血红蛋白对分析无害。得到的基本上无血红蛋白的样品通过毛细力或重力被转移至下面的试验垫片上。在试验垫片上，样品引发级联反应，产生有色的染料，其浓度和样品中的分析物浓度成比例，用光度计可直接确定。图4描述了使用葡萄糖氧化酶和PMS的葡萄糖的反应。
图5描述了有和没有亚硝酸盐的血样品在时间范围内的光密度的变化，都具有60％血细胞比容并且都含有0-100mg/dL的葡萄糖。上图显示了在35℃下的结果。下图显示了在室温(RT)下的结果。在每个情况下，亚硝酸盐的浓度是5g/dL。在没有亚硝酸盐的情况下，血红蛋白减少了四氮唑以形成连续增加的染料浓度并相应增加了光密度。通过除去血红蛋白(通过氧化)，亚硝酸盐限制了颜色的形成以致结果仅由样品中的葡萄糖得到。制备用于产生图中所描述的数据的两层条在下面的实施例1中有述。
图6显示了亚硝酸盐对单层条的葡萄糖/葡萄糖氧化酶系统中的颜色形成反应的影响。都具有60％血细胞比容的血样品含有0或200mg/dL葡萄糖和0或200mg/dL的亚硝酸盐。在室温下操作样品。图显示了本系统在室温下是有效的，血细胞比容高达60％。制备使用的单层条在下面的实施例2中有述。
下面的实施例说明了本发明的优选实施方案。在实施例1中，使用两层条，分析物是葡萄糖，酶是葡萄糖酶。在实施例2中，使用单层条。如前所述，分析物是葡萄糖，酶是葡萄糖酶。可容易地改进该组合物以应用于以前所列的其它分折物-酶的组合。(参见，例如Tietz Texbook of Clinical Chemistry，第二版，由C.Burtis等人编辑，W.B.Saunders公司，Philadelphia，PA，第976-978和1174-1175页。)实施例不意在对本发明有任何限制。
                             实施例1
将得自Pall公司(East Hills，NY)的0.8μm尼龙膜浸在表1的试剂中直至饱和。用玻璃棒轻轻地刮掉过量的试剂。将得到的膜悬挂在56℃的烘箱中干燥10分钟。将Porex(0.6mm厚)浸泡在表2的亚硝酸盐溶液中，然后悬挂在100℃的烘箱中干燥10小时。最后，膜在聚酯料(0.4mm Melenex聚酯，得自ICIAmerica，Wilmington，DE)和浸满亚硝酸盐的Porex之间层压。
                             实施例2
重复实施例1的步骤，除了第一次浸泡是在表3的试剂中，没有第二次浸泡，因为不需要Porex。
表1葡萄糖试验垫片的试剂
组分   用量
水   100ml
(2-[吗啉代]乙磺酸)钠盐MES(MW 217.2，Sigma，St.Louis，MO，USA)通过加6M HCl调节pH至5-7   2.2g
Tetonic 1307(BASF Corporation，Moun Olive，New Jersey，USA)   1-3g
PSSA，聚苯乙烯磺酸钠盐(MW70000，Polysciences，Inc.，Warrington，PA，USA)   2-4g
Crotein(Croda Inc.，Parsippany，NJ，USA)   2-4g
甘露糖醇(MW182，Sigma，St.Louis，MO，USA)   1-10g
吩嗪硫酸甲酯(PMS，MW306.34，Sigma，St.Louis，MO，USA)   30-300mg
WST-5(MW1331.37，Dojindo)   0.8-4g
葡萄糖氧化酶(GO，TOYOBO)   100-1000KU
表2亚硝酸盐试剂
组分     用量
10mM磷酸盐缓冲液，pH7.4(P-3813，Sigma，St.Louis，MO，USA)     70ml
乙醇     30ml
亚硝酸钠(MW69，Aldrich Chemicals，Milwaukee，WI，USA)     5g
聚乙烯吡咯烷酮(polyvinylpyrrodine)(MW40000，Sigma，St.Louis，MO，USA)     200mg
表3葡萄糖试验垫片的试剂
组分   用量
水   100ml
(2-[吗啉代]乙磺酸)钠盐MES(MW217.2，Sigma，St.Louis，MO，USA)   2.2g
Gantrez*6％   20ml
通过添加50％NaOH，调pH至5.5-7
Triton X-305(BASF Corporation，Moun Olive，New Jersey，USA)  0.5-2g
甘露糖醇(MW182，Sigma，St.Louis，MO，USA)   1-10g
亚硝酸钠(MW69，Aldrich Chemicals，Milwaukee，WI，USA)   1-5g
WST-5(MW1331.37，Dojindo)   0.8-4g
氯化镁(MW203，Sigma，St.Louis，MO，USA)   3-5g
吩嗪乙硫酸盐(PES，MW334.4，Sigma，St.Louis，MO，USA)   100-1000mg
葡萄糖氧化酶(GO，TOYOBO)   100-1000KU
*Gantrez AN-139(聚甲基乙烯基醚-alt-马来酸酐，MW1080000，Cat#41632-0，Aldrich Chemicals，Milwaukee，WI，USA)使6％Gantrez在水中，加热至95℃，少于45分钟，得到易于使用的Gantrez6％。

Claims (22)

1.一种测量含血红蛋白的生物流体中的分析物浓度的试剂，包含
a)具有与之结合的黄素和对分析物具有特异性的依赖黄素的酶，
b)四氮唑染料前体，
c)电子转移剂，和
d)亚硝酸盐。
2.权利要求1的试剂，其中分析物是葡萄糖，酶是葡萄糖氧化酶。
3.权利要求1的试剂，其中染料前体是水溶性的。
4.权利要求1的试剂，其中电子转移剂包含吩嗪硫酸甲酯(PMS)或其同系物。
5.权利要求1的试剂，进一步包含二价金属稳定剂。
6.一种测量含血红蛋白的生物流体中的分析物浓度的试剂，包含
a)对分析物具有特异性的和没有与之相结合的黄素的依赖黄素的酶，
b)黄素单核苷酸(FMN)或黄素腺嘌呤二核苷酸(FAD)，
c)四氮唑染料前体，
d)电子转移剂，和
e)亚硝酸盐。
7.权利要求6的试剂，其中染料前体是水溶性的。
8.权利要求6的试剂，其中电子转移剂包含(PMS)或其同系物。
9.权利要求6的试剂，进一步包含二价金属稳定剂。
10.一种测量含血红蛋白的生物流体中的分析物浓度的干试剂条，包括一载体层，其上是具有权利要求1的试剂的涂层的试验垫片。
11.一种测量含血红蛋白的生物流体中的分析物浓度的干试剂条，包括一载体层，其上是具有权利要求6的试剂的涂层的试验垫片。
12.权利要求10的条，其中试验垫片具有带正电荷的表面。
13.权利要求10的条，其中试验垫片含有聚酰胺。
14.权利要求10的条，进一步含有覆盖在试验垫片上的吸湿的上层。
15.一种测量含血红蛋白的生物流体中的分析物浓度的干试剂条，包括一载体层，其上是试验垫片，一上层覆盖在试验垫片上，其中权利要求1的试剂的第一部分在试验垫片上，第二部分试剂在载体和/或上层上。
16.一种测量含血红蛋白的生物流体中的分析物浓度的干试剂条，包括一载体层，其上是试验垫片，一上层覆盖在试验垫片上，其中权利要求6的试剂的第一部分在试验垫片上，第二部分试剂在载体和/或上层上。
17.权利要求15的条，其中上层是吸湿性的。
18.权利要求15的条，进一步包括上层与试验垫片之间的间隔和通道，提供上层和垫片之间的毛细路径。
19.权利要求15的条，其中分析物是葡萄糖，酶是葡萄糖氧化酶。
20.权利要求15的条，其中四氮唑染料前体是2，2′-二苯并噻唑基-5，5′-双[4-二(2-磺基乙基)氨基甲酰基苯基]-3，3′-(3，3′-二甲氧基-4，4′-亚联苯基)二四氮唑二钠盐(WST-5)。
21.一种测量含血红蛋白的生物流体中的葡萄糖浓度的干试剂试验条，包括
a)一载体层，
b)在载体层上有一含有涂层的试验垫片，该涂层含有
i)具有与之结合的黄素的葡萄糖氧化酶，
ii)四氮唑染料前体，和
iii)PMS或其同系物，和
c)在试验垫片上，有一吸湿性的上层，用亚硝酸盐涂敷该层。
22.一种测量含血红蛋白的生物流体中的葡萄糖浓度的干试剂试验条，包括
a)一载体层，
b)在载体层上有一含有涂层的试验垫片，该涂层含有
i)没有与之相结合的黄素的依赖黄素的酶，
ii)FMN或FAD，
iii)四氮唑染料前体，和
iv)PMS或其同系物，和
c)在试验垫片上的用亚硝酸盐涂敷的吸湿顶层。
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