CN100439513C - 含有非再生酶-辅酶复合体的方法和试剂体系 - Google Patents
含有非再生酶-辅酶复合体的方法和试剂体系 Download PDFInfo
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Abstract
本发明涉及一种通过酶促反应方法检测样品中分析物的方法和试剂体系，包括用酶-辅酶复合体作为化学计量反应配偶体来检测存在于样品中的分析物。
Description
本发明涉及一种通过酶促反应检测样品中分析物的方法和试剂体系，包括酶-辅酶复合体作为非再生、特别是化学计量量的反应物检测存在于样品中的分析物的用途。
已知通过酶促方法检测分析物，如血中的葡萄糖。这需要待分析的分析物与合适的催化量的酶和辅酶接触。辅酶在还原作用或氧化作用中所产生的氧化还原当量传递给介质，在随后的步骤中利用电化学或光度学方法检测介质。有一个校准提供了检测值和待测分析物浓度之间的直接关系。
Sierra等(Anal.Chem.69(1997)，1471-1476)描述了一种基于葡萄糖氧化酶内在荧光特性的血葡萄糖检测方法。在该方法中，使用了催化量的酶和它的辅酶FAD，氧化还原当量被传递给介质氧。
Narayanswamy等(Aanlytical letters21(7)(1988)，1165-1175)描述了一种用于葡萄糖检测的葡萄糖脱氢酶和NAD荧光测定方法。在此情况下所使用的酶是催化，即非化学计量的量。用荧光测定方法检测溶液中游离的NADH。
在现有技术检测体系中，需要电化学活性物质(介质)来检测待测的分析物，这是非直接的，即需要通过一系列的化学反应。在此情况下，通常需要对所用物质的浓度作出复杂的调整以尽量提高反应速度。而且，在危险是较长的贮藏阶段，所需的电化学活性物质是不稳定的。
而且，相对于酶-辅酶体系，通常不得不使用过量的介质。辅酶具有高的反应活性，因此介质的分解即使是很小的量，如小于1％或暴露于外部物质，如物质从包装材料向外的挥发，也会导致酶活性的急剧下降。这在分析物检测中会导致错误信号的产生。而且还有一个缺点是，分析物的检测时间通常至少要几秒，对于葡萄糖例如需要大于4秒，并且所需的样品量是大量的，如大于0.5μl。
本发明的目的是至少在一定程度上避免上述现有技术中的缺点。特别是提供一种非敏感和快速的用于分析物酶促检测的方法，使得即使不存在介质或/和指示剂的情况下仍然能够获得可靠的测定结果。
本发明的目的是通过使用酶-辅酶复合体作为化学计量量的反应物，而不是象通常那样作为催化剂来实现的。分析物的检测仅需需要唯一的反应步骤，因此非常快速。在此不再需要介质和指示剂以及复杂的反应混合物，因此也不再具有低稳定性和对干扰的高度敏感性。
因此，本发明的一个方面是提供了一种通过酶促反应检测样品中分析物的方法，包含以下步骤；(a)使样品与包含酶-辅酶复合体的检测试剂接触，其中不发生辅酶的再生，和(b)通过酶-辅酶复合体的变化检测分析物的反应。
本发明的另一方面是提供一种用于检测样品中分析物的试剂体系，包含：(a)包含酶-辅酶复合体的检测试剂，其中不发生辅酶的再生，和(b)容纳检测试剂的载体。
本发明使分析物在非常短的时间内，优选≤5秒，特别优选≤1秒，最优选≤0.1秒，进行简单的定性或定量检测成为可能。反应是在检测过程中不发生辅酶再生的条件下进行的。此外，分子酶-辅酶复合体可仅和一分子的分析物反应。因此，有利的是，反应应当在不存在介质或其它能够导致辅酶再生的物质的条件下进行。
根据所描述的试验设计，检测试剂包含足够量的用于定性或/和定量检测分析物的酶-辅酶复合体。尤其是对于分析物的定量检测，所使用的酶-辅酶复合体的量应当使酶-辅酶复合体的反应分子数目与存在于样品中的分析物的浓度相关联。特别优选所使用的酶-辅酶复合体的量相对于样品中分析物应当是至少化学计量的量，优选相对于分析物是过量的化学计量的量。在这里，“至少化学计量的量”意味着相对于酶-辅酶复合体分子的数目，需要对样品大小作出调整，使得根据预期的样品中分析物的浓度，与分析物反应的酶-辅酶复合体的分子数目与存在于样品中的分析物浓度相关联。“化学计量的量”优选使得指酶-辅酶复合体的分子数目与研究样品中预期的分析物分子最大数目相一致。
本方法和检测体系可以使用最少量的样品，例如，≤1μl的样品量，特别是≤0.1μl的样品量。任选地样品可以在与检测试剂接触前进行稀释。
本发明的方法和检测体系适合检测任何分析物，例如体液参数，体液如血、血清、血浆或尿，也可以是废水或食品。该方法可以在湿态条件下进行如在试管中，或者在干态下有合适反应物载体的条件下进行。
被检测的分析物可以是任何能够与酶-辅酶复合体进行反应，特别是氧化还原反应的生物或化学物质，例如，葡萄糖、乳酸、苹果酸、甘油、醇、胆固醇、甘油三脂，抗坏血酸、半胱氨酸、谷胱甘肽、肽等。
酶促反应优选是氧化还原反应，其中酶-辅酶复合体中的辅酶被还原或氧化。在这类反应中，优选使用的酶是氧化还原酶。特别优选使用的酶是脱氢酶，例如选自葡萄糖脱氢酶(E.C.1.1.1.47)、乳酸脱氢酶(E.C.1.1.127，1.1.1.28)、苹果酸脱氢酶(E.C.1.1.1.37)、甘油脱氢酶(E.C.1.1.1.6)、醇脱氢酶(E.C.1.1.1.1)或氨基酸脱氢酶，如L-氨基酸脱氢酶(E.C.1.4.1.5)。其它适合的酶是氧化酶，例如，葡萄糖氧化酶(E.C.1.1.3.4)或胆固醇氧化酶(E.C.1.1.3.6)。
用于本发明目的的辅酶优选是有机分子，其与酶共价或非共价连接，并且通过分析物的转化而发生变化，如被氧化或被还原。优选的辅酶例子有黄素、烟碱和苯醌衍生物，如核黄素衍生物，如FAD、FADH2、FMN、FMNH2等，烟碱核苷酸衍生物，如NAD+、NADH/H+、NADP+、NADPH/H+等，或泛醌，如辅酶Q、PQQ等。
辅酶通过与分析物反应而发生的变化，原则上可以通过任何方法得到检测。原则上可以使用现有技术中已知的所有检测酶促反应的方法。然而，优选使用光学方法来检测辅酶的变化。光学检测方法包括如测定吸收、荧光、圆二色性(CD)、旋光色散(ORD)、折射测定法等。特别优选通过测定荧光来检测辅酶的变化。荧光测定是高度灵敏的并且即使在微型化体系下分析物的浓度很低仍然能够检测。
本发明的方法和检测体系可以包含液体检测，在此情况下试剂是以例如溶液或悬浮在水或非水液体中、或以粉末或冻干粉的形式存在。但是本发明的方法和检测体系优选包含干态检测，在此情况下试剂被放置到载体上。载体包括例如含有吸附剂或/和可膨胀材料的、可以被待检测样品液体湿润的检测条。
在一个特别优选实施方式中，所使用的检测试剂是凝胶基质，其中嵌入酶-辅酶复合体。优选凝胶基质层厚≤50μm，特别≤5μm，并且被放置到载体上，载体例如至少在一定程度上是光学透明的。凝胶基质可以是含有一种或多种可溶性聚合物的基质，同已知的干态检测体系相同(如AccuChek Active)，并且可被刀割下并干燥制备。基质优选基于有可光聚合结构特性的物质，如丙烯酸单体如丙烯酰胺或/和丙烯酸酯、如聚乙二醇二丙烯酸酯、或乙烯芳香族单体如4-乙烯苯磺酸、或其组合的聚合物。该类型凝胶基质可以通过如下方式制备，将含有酶、可光聚合特性单体和任选的辅酶的试剂、光引发剂或/和不反应成分的液体涂覆到至少部分透明的载体如塑料片上，并且例如用UV光从背面照射，使得一种单体或多种单体在载体上发生聚合并达到预定的层厚。层厚可以通过在试剂中加入吸附剂或/和通过调节照射的时间和强度来控制。可以在聚合后去掉过量的液体试剂，并再利用(参看例如图2)。
另一方面，凝胶基质还可以通过通常的涂覆步骤来制备，其中将液体试剂涂覆到载体上，使用合适的方法如使用刮刀使之达到所需要的厚度，然后使之完全聚合。
通过聚合或嵌入包含到基质后，酶处于被保护的微环境中。如果聚合物的凝胶基质是充分交联的，则酶分子处于不可移动状态。然而，低分子量物质或葡萄糖或其它分析物或其辅酶则可以在聚合物网状结构中自由扩散。
酶既可以通过聚合作用与其辅酶共同包含于基质中，也可以在聚合作用后将基质与辅酶溶液接触，使得形成合适的酶-辅酶复合体。凝胶基质中酶的浓度优选足够与待测分析物进行化学计量反应，并且能够进行被反应改变了的辅酶的直接检测。反应仅由唯一的催化反应组成，如氧化还原反应，其可以在毫秒或微秒的时间间隔内完成。被反应改变了的辅酶通过结合到酶的活性中心而得到最适的保护，使其免受干扰影响，并且任选地还可以将其嵌入凝胶基质中。
以下通过附图和实施例详细描述本发明。
附图
图1表示本发明检测体系第一种实施方式。试剂层(2)，如含有酶-辅酶复合体的凝胶基质，涂布到透明的载体(1)上。酶-辅酶复合体是以一种在分析物检测期间使辅酶不能发生再生的形式存在。样品(3)如血，加到试剂层上。包含在样品(3)中的分析物和包含在试剂层(2)中的酶-辅酶复合体之间发生酶促反应通过光学方法进行检测。从光源(4)发出的光，如激光或LED，从后面(穿透载体)射到试剂层(2)。通过检测器(5)检测从样品反射回来的吸收光或荧光。任选地，特别是对于荧光检测，在检测器的前面放上光学过滤元件(6)，目的是防止激发荧光的泄漏。
图2表示本发明检测体系的制备。将液体试剂(12)如第一个位置(13)涂到光透明的载体(11)上，如塑料片。从光源(14)发出的光从后面透过载体(11)照射到位于第二位置的液体试剂(12)。同时，沿着(15)所示的箭头方向移动载体。从而在载体(11)上直接形成聚合了的试剂层(16)。在聚合层(16)上有过量的液体试剂。聚合了的反应物层(16)的厚度可以通过调节试剂的组成、光束的强度和照射时间和通过载体(11)的性质来进行控制。
图3表示从后面的荧光传感器的实施方式。聚合了的试剂层，例如通过图2所示的连续步骤制备而成，它可以被切下，利用已知技术放置到载体(21)。将样品放置到向上的一面，从光源射出的激发光(23)如UV从下面射入。分析物与试剂层(22)的酶-辅酶复合体反应产生荧光(24)如蓝光，并用检测器对其进行检测。
将多种试剂(相同或不同)放置到载体上也是可以的。图4显示了其中一个圆盘形式的实施方式。多个试剂点(32)排列在光透明的载体(31)上。
图5A和5B显示了在CCD摄像机下本发明检测体系的荧光(葡萄糖脱氢酶和NAD+)与葡萄糖浓度增加的关系。
实施例
实施例1：在试管中葡萄糖脱氢酶(GlucDH)/NAD+体系下葡萄糖化学计量量的检测
100mg/ml GlucDH溶解于pH7的缓冲溶液并与适量的NAD+混合。随着葡萄糖量的增加，将会在紫外灯(激发波长为366nm)下观察到荧光强度的增加(图5A和5B)。
没有葡萄糖的酶体系的溶液不发出荧光，并且只有葡萄糖和NAD+的溶液也不发出荧光。
实施例2：在聚合物薄膜上在GlucDH/NAD+体系下进行的葡萄糖检测
在塑料试管中混合如下物质的悬浮液
配方1
| 物质 | 量(g) | 重量(％) |
| 丙烯酰胺 | 2.5 | 22.02 |
| 亚甲基双丙烯酰胺 | 0.7 | 6.17 |
| 2，2-二甲氧基-2苯基苯乙酮 | 0.05 | 0.44 |
| 甘油 | 5 | 44.05 |
| 甲基丙烯酸羟乙酯 | 1.4 | 12.33 |
| 甲基丙烯酸甲酯 | 0.4 | 3.52 |
| Crodasinic O溶液，pH8，0.3g/1000ml | 1 | 8.81 |
| N，N’-(1，2-二羟亚乙基)双丙烯酰胺 | 0.3 | 2.64 |
| 总量 | 11.35 | 100 |
0.5ml的悬浮液与0.5ml的GlucDH(100mg/ml)溶液混合，混合物在超声水浴锅中均化去掉气泡。
澄清的溶液倒入电晕处理的聚碳酸酯片上，其厚度125mm，并用常用的照射装置(Isel UV照射仪2)照射20分钟。塑料片短暂地用水冲洗并随后在空气中干燥。
制得的层的厚度＜2μm。制备新鲜的葡萄糖/NAD+溶液(GKL-3溶液，300mg/dl的葡萄糖，1ml/6.4mg的NAD+)并倒到薄膜上。在紫外灯下立即可看到强烈的荧光。
实施例3：加紫外吸收剂对层厚的影响
制备更能证明试验结果的包含蓝色染料(最大吸收波长≈650nm)的聚合物层(配方2)。在进一步的试验中，在最初的配方中掺入了黄色染料作为UV吸收剂(配方3)。
配方2
| 物质 | 量 | 重量(％) |
| 丙烯酰胺 | 37.5g(0.53mol) | 25.78 |
| 聚乙二醇二丙烯酸酯，Mw≈575g/mol | 52.5g(约0.96mol) | 36.10 |
| Crodasinic O(0.3g/l l)溶液 | 50g | 34.38 |
| 4-乙烯基苯磺酸 | 5g | 3.44 |
| 2，2-二甲氧基-2-苯基苯乙酮光引发剂 | 350mg | 0.24 |
| 新亚甲蓝N | 100mg | 0.06 |
| 总量 | 145.45g | 100 |
搅拌混合物，并经过超声浴处理均化，用吸管分布到140μm的Pokalon片上(电晕处理，步骤4)并用紫外照射仪(Actina U4，W.Lemmen GmbH)照射1分钟。
用螺旋测定仪(Mikrometerschraube)测量制得的层的厚度，结果为2405μm。
配方3
| 物质 | 量 | 重量(％) |
| 配方2 | 1ml | 约99.99 |
| 黄色媒染剂7(No.686)(紫外吸收剂) | 0.0001g | 0.001 |
| 总量 | 约1.0001g | 100 |
将混合物如上所述分布到薄片上并随后聚合。用螺旋测定仪测量制得的层的厚度，结果为79.3μm。
试验结果表明是可以影响层的厚度的。其它条件相同的条件下，没有紫外吸收剂的情况下层厚240.5μm；在有紫外吸收剂(黄色媒染剂7)的情况下层厚仅为79.3μm。
Claims (23)
1.通过酶促反应检测样品中分析物的方法，包含如下步骤：(a)在不发生辅酶再生的条件下，使样品与包含酶-辅酶复合体的检测试剂接触，其中酶是脱氢酶，和(b)通过酶-辅酶复合体的变化检测分析物的反应。
2.根据权利要求1所述的方法，其特征在于所述脱氢酶选自葡萄糖脱氢酶(E.C.1.1.1.47)、乳酸脱氢酶(E.C.1.1.1.27，1.1.1.28)、苹果酸脱氢酶(E.C.1.1.1.37)、甘油脱氢酶(E.C.1.1.1.6)、醇脱氢酶(E.C.1.1.1.1)或氨基酸脱氢酶。
3.根据权利要求1所述的方法，其特征在于所使用的辅酶选自黄素、烟碱和苯醌衍生物。
4.根据权利要求3所述的方法，其特征在于辅酶选自FAD、FADH2、FMN、FMNH2、辅酶Q、PQQ、NAD+、NADH/H+、NADP+和NADPH/H+。
5.根据权利要求1-4中任一项所述的方法，其特征在于通过光学方法检测辅酶的变化。
6.根据权利要求5所述的方法，其特征在于通过测定吸收、荧光、CD、ORD或折射来检测辅酶的变化。
7.根据权利要求6所述的方法，其特征在于通过测定荧光来检测辅酶的变化。
8.根据权利要求1-4中任一项所述的方法，其特征在于使用其中具有嵌入的酶-辅酶复合体的凝胶基质作为检测试剂。
9.根据权利要求8所述的方法，其特征在于凝胶基质的层厚≤50μm。
10.根据权利要求9所述的方法，其特征在于凝胶基质的层厚≤5μm。
11.根据权利要求8所述的方法，其特征在于使用以可光聚合物质为基础的凝胶基质。
12.根据权利要求1-4中任一项所述的方法，其特征在于检测体液中的分析物。
13.根据权利要求12所述的方法，其特征在于进行血中葡萄糖的检测。
14.根据权利要求1-4中任一项所述的方法，其特征在于分析物的反应时间≤5秒。
15.根据权利要求1-4中任一项所述的方法，其特征在于反应是在不存在可与辅酶发生反应的介质的情况下进行的。
16.根据权利要求1-4中任一项所述的方法，其特征在于相对于分析物，使用的酶-辅酶复合体作为化学计量量的反应物。
17.根据权利要求2所述的方法，其特征在于所述氨基酸脱氢酶为L-氨基酸脱氢酶(E.C.1.4.1.5)。
18.一种用于检测样品中分析物的试剂体系，包含：(a)包含以不发生辅酶再生的形式存在的酶-辅酶复合体的检测试剂，其中使用的酶是脱氢酶，和(b)容纳检测试剂的载体。
19.根据权利要求18所述的试剂体系，其特征在于酶-辅酶复合体嵌入凝胶基质中。
20.根据权利要求18或19所述的试剂体系，其特征在于载体至少部分地是光学透明的。
21.根据权利要求18或19所述的试剂体系，其特征在于载体包含平的结构。
22.根据权利要求18或19所述的试剂体系，其特征在于载体包含多个不同的检测试剂。
23.根据权利要求18-22任一项所述的试剂体系在权利要求1-17任一项所述的方法中的应用。
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| DE102013104906B4 (de) * | 2013-05-13 | 2015-06-25 | Brandenburgische Technische Universität Cottbus-Senftenberg | Wasserfreie Immobilisierung von Enzymen |
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