CN1914331A - 作为生物传感器的内部参照的可氧化种类和使用方法 - Google Patents
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Abstract
提供了用于测定样品中物质的存在或者量的生物传感器(102)和该生物传感器(102)的使用方法。用于接受待分析的用户样品的生物传感器(102)包括用于与分析物发生电化学反应的混合物。该混合物包括酶、介体和作为内部参照的可氧化的种类。
Description
发明领域
本发明总体上涉及生物传感器,且更具体地,涉及新的和改进的生物传感器,其包括作为内部参照的可氧化种类,还涉及该生物传感器的使用方法,以用于测定样品中物质的存在或者量。
现有技术描述
体液中分析物的定量测定在诊断和某些生理学异常的维持中非常重要。例如,应该监控某些个体中的乳酸、胆固醇和胆红素。具体地,体液中葡萄糖的测定对于糖尿病个体非常重要,这些个体必须经常检查他们的体液中的葡萄糖水平作为调节他们的饮食中葡萄糖摄入的手段。尽管本公开内容的剩下内容将涉及葡萄糖的测定,但是将理解本发明的新的和改进的传感器元件和使用方法可以在选择合适的酶后测定其他分析物。
流体中分析物浓度的测定方法可以基于该分析物和对该分析物特异的酶和维持该酶处于其最初氧化状态的介体之间的电化学反应。适宜的氧化还原酶包括氧化酶、脱氢酶、过氧化氢酶和过氧化物酶。例如,对于分析物是葡萄糖的情况,与葡萄糖氧化酶和氧的反应通过如下反应式表示:
在反应式(A)表示的反应的初始步骤中,测试样品中存在的葡萄糖将酶(Eox),如酶的氧化的黄素腺嘌呤二核苷酸(FAD)中心转化成它的还原形式(Ered),例如,(FADH2)。因为这些氧化还原中心在酶分子内基本上是电绝缘的,所以在不存在不可接受的高细胞电压时,不发生任何可测量程度的向常规电极表面的直接电子转移。该系统的改进涉及在电极和酶之间使用非生理氧化还原偶联以在(FADH2)和电极之间往返运输电子。这通过下面的方案表示,其中氧化还原偶合剂(通常称作介体)由M表示:
在该方案中,GO(FAD)代表葡萄糖氧化酶的氧化形式,GO(FADH2)表示它的还原形式。介导种类Mox/Mred将电子从还原的酶往返运输到电极,从而氧化该酶,导致它原位再生。
授予Genshaw等人并且转让给本受让人的美国专利号5,620,579和5,653,863公开了测定流体测试样品中分析物的浓度的装置和方法,其通过将流体测试样品应用于工作电极的表面来实施,所述工作电极电化学地连接对电极,并且其表面含有组合物,该组合物包含对所述分析物特异的酶。响应于分析物和酶之间的反应,介体被还原。在测定分析物的浓度之前在电极之间应用氧化电位以将至少一部分介体返回到它的氧化形式,从而增加分析物测定的准确性。在该最初应用的电位后,将电路切换到断路或者显著减小电流的电位,以使工作电极处电化学电位率最小化。在电极之间应用第二种电位并测量流体测试样品中产生的电流以测定分析物浓度。任选地,通过算法进一步增强分析物测定的准确性。
发明概述
本发明的重要方面是提供新的和改进的生物传感器,其用于测定样品中物质的存在或者量,所述生物传感器包括作为内部参照的可氧化的种类,还涉及该生物传感器的使用方法。
简言之,提供了用于测定样品中物质的存在或者量的生物传感器和该生物传感器的使用方法。用于接收待分析的用户样品的生物传感器包括用于与分析物发生电化学反应的混合物。该混合物包括酶、介体和作为内部参照的可氧化的种类。
将内部参照定义为可氧化的种类,其在一个实施方案中可以进一步定义为可逆氧化还原偶的还原形式,其具有与介体相等或者更高的氧化还原电位。内部参照对于氧化两种种类的工作电位加性地增加响应电流,且对于分析物是葡萄糖的情况,总的响应电流通过如下表示:
I总的=I内部参照+I葡萄糖
I内部参照∝(内部参照)并且I葡萄糖∝(葡萄糖)其中I内部参照是由于内部参照造成的总响应电流的部分,而I葡萄糖是由于介体氧化造成的,其与葡萄糖浓度成比例。
根据本发明的特征,内部参照可以是相同的介体种类或者具有比介体更高的氧化还原电位的可氧化的种类。从而,对于具有仅氧化介体的低工作电位的生物传感器,电流I内部参照将为0。然而,对于具有氧化两种种类的更高的工作电位的生物传感器,总响应电流将是由于内部参照导致的部分和由于葡萄糖导致的部分的总和。因为内部参照浓度是固定的,所以传感器的校准斜率将仅仅取决于葡萄糖的传感器响应而截距将取决于所加的内部参照的量。换句话说,内部参照将仅仅偏移截距并且不改变校准斜率。从而,内部参照的概念提供了制造葡萄糖生物传感器的新的且不同的方法。
附图简述
从下面附图中阐明的本发明的优选实施方案的详细描述,可以最好地理解本发明以及上面和其他目的和优点。附图中:
图1A是生物传感器计量器的方块图表示,该生物传感器计量器包括具有根据本发明的内部参照的生物传感器;
图1B、1C和1D是分别阐明使用本发明的图1的生物传感器的操作方法的图解。
图2A、2B和2C显示了用亚铁氰化物作为本发明图1的生物传感器的内部参照,在0mg/dL葡萄糖的全血样品中,基于MLB的葡萄糖生物传感器的三个循环伏安图。
图3是阐明了在不同电压工作电位下,本发明的图1的生物传感器的线性响应的图。
图4是阐明了使用本发明的图1的生物传感器,用10%印刷的铁氰化物作为对电极,所加入的内部参照对总体伏安电流的影响的图。
图5A和5B是阐明了用Ag/AgCl作为对电极,本发明图1的基于MLB的生物传感器的线性响应和增加的内部参照导致的增加的截距的图。
图6A和6B是阐明了用10%铁氰化物作为对电极,本发明图1的基于MLB的生物传感器的线性响应和增加的内部参照导致的增加的截距的图。
图7是阐明了用10%铁氰化物作为对电极,本发明图1的DEX生物传感器的校准截距与增加的内部参照的线性关系的图。
图8A和8B阐明了本发明的图1的生物传感器的来自对照试剂墨水和向20%铁氰化物的试剂混合物中加入0.1%亚铁氰化物的试剂墨水残留的亚铁氰化物的流动注射分析(FIA)产生的信号与参照比。
优选实施方案的详细描述
本发明涉及用于测定样品中物质的存在或者量的电化学生物传感器。该生物传感器包括含有工作电极和对电极的传感器条,工作电极和对电极的每一种都用例如,单独的试剂层至少部分覆盖。工作电极上的试剂层包括例如,通过氧化还原反应与分析物相互作用的酶并且还包括介体,其为氧化还原偶的氧化形式。本发明的生物传感器包括工作电极上试剂层中的内部参照或者还原形式的介体。将内部参照定义为可氧化的种类,其在一个实施方案中可以进一步定义为可逆氧化还原偶的还原形式,其具有与介体相等或者更高的氧化还原电位。在试剂层中提供了固定量的内部参照。包括内部参照或者加入量的还原形式的介体的本发明的生物传感器提供了改进,因为内部参照用于通过热力学性质使校准截距稳定同时维持校准斜率。
许多化合物由于它们能够从还原的酶接受电子并将它们转移到电极而用作介体。介体的必要性质是在传感器使用前能够在电极表面上存在的条件下保持氧化状态。更古老的介体是氧化形式的有机金属化合物、有机分子、过渡金属配位络合物。介体的特定实例是六氰基高铁酸钾(III),也称作铁氰化物。
如在下面说明书和权利要求书中使用的,术语生物传感器指分析装置或者仪器的电化学传感器条或者传感器元件,其选择性地响应适宜样品中的分析物并将它们的浓度转化成电信号。生物传感器直接产生电信号,从而方便了简单的仪器设计。同样,生物传感器提供了低廉的材料成本的优点,因为将薄层化学药品沉积在电极上并且很少有材料浪费。
将术语“样品”定义为含有未知量的目的分析物的组合物。通常,用于电化学分析的样品是液体形式,且优选地该样品为水性混合物。样品可以是生物样品,如血液、尿或者唾液。样品可以是生物样品的衍生物,例如,提取物、稀释液、滤液或者重构的沉淀物。
将术语“分析物”定义为样品中的物质,其存在或者量待测定。分析期间,分析物与存在的氧化还原酶相互作用,并且可以是氧化还原酶的底物、辅酶或者影响氧化还原酶和其底物的相互作用的另一种物质。
术语“氧化还原酶”定义为促进底物的氧化或者还原的任何酶。术语氧化还原酶包括“氧化酶”,其促进氧化反应,其中分子氧是电子受体;“还原酶”,其促进还原反应,其中分析物被还原并且分子氧不是分析物;和“脱氢酶”,其促进氧化反应,其中分子氧不是电子受体。见,例如,Oxford Dictionary of Biochemistry andMolecular Biology,修订本,A.D.Smith,Ed.,New York:OxfordUniversity Press(1997)pp.161,476,477,和560。
术语“氧化还原”反应定义为两种种类之间的化学反应,其涉及至少一个电子从一个种类向另一种类的转移。该类型的反应也称作“氧化还原反应”。该反应的氧化部分涉及一个种类丧失至少一个电子,且还原部分涉及向另一种类加入至少一个电子。被氧化的种类的离子电荷以等于转移的电子数目的量计电正性更强。同样的,被还原的种类的离子电荷以等于转移的电子数目的量计电正性更弱。
将术语“氧化值”定义为化学种类,例如原子的形式离子电荷。较高的氧化值,如(III),电正性更强,而较低的氧化值,如(II),电正性更弱。中性种类具有O离子电荷。种类的氧化导致该种类的氧化值的增加,而种类的还原导致该种类的氧化值的减少。
术语“氧化还原对”定义为具有不同氧化值的两种化学物质。具有更高氧化值的种类的还原产生具有较低氧化值的种类。备选地,具有较低氧化值的种类的氧化产生具有较高氧化值的种类。
术语“可氧化的种类”定义为具有较低氧化值的氧化还原对的种类,并且其从而能够氧化成具有较高氧化值的种类。同样的,术语“可还原的种类”定义为具有较高氧化值的氧化还原对的种类,并且其从而能够还原成具有较低氧化值的种类。
术语“有机过渡金属络合物”也称作“OTM络合物”,定义为这样的络合物,其中过渡金属通过σ键(结合过渡金属的碳原子σ上-1的形式电荷)或者π键(结合过渡金属的碳原子π上的O形式电荷)结合至少一个碳原子。例如,二茂铁是具有两个环戊二烯基(Cp)环的OTM络合物,每个环通过其5个碳原子通过2个π键和1个σ键结合铁中心。OTM络合物的另一个实例是铁氰化物(III)和其还原的亚铁氰化物(II)对应物,其中6个氰基配体(6个配体的每个上-1的形式电荷)通过氰基的碳原子σ结合到铁中心。
术语“配位络合物”定义为具有确定的配位几何形状,例如,八面体或者正方形平面几何形状的络合物。与OTM络合物(其通过它们的键合定义)不同,配位络合物通过它们的几何形状定义。从而,配位络合物可以是OTM络合物(如前面提到的铁氰化物),或者是这样的络合物,其中非碳的非金属原子,如杂原子(包括氮、硫、氧和磷)配价地结合到过渡金属中心。例如,钌六胺或者六氨基钌酸盐(II)/(III)是具有确定的八面体几何形状的配位络合物,其中六个NH3配体(6个配体的每个上O形式电荷)配位地结合钌中心。铁氰化物也是具有八面体几何形状的配位络合物的实例。关于有机过渡金属络合物、配位络合物和过渡金属键合的更完全的讨论可以见Collman等人,Principles and Applications of Organotransition MetalChemistry(1987)和Miessler&Tarr,Inorganic Chemistry(1991)。
术语“介体”定义为可以氧化或还原并且可以在第一种物质和第二种物质之间转移一个或多个电子的物质。介体是电化学分析中的试剂并且不是目的分析物。在简化系统中,在氧化还原酶通过与适宜底物接触被还原或者氧化后,介体经历与氧化还原酶的氧化还原反应。该氧化或者还原的介体然后在电极经历相反反应并再生为其最初的氧化值。
术语“电活性有机分子”定义为不含有金属并且能够经历氧化或者还原反应的有机分子。电化学活性有机分子可以作为氧化还原种类和作为介体。电化学活性有机分子的实例包括辅酶吡咯并喹啉醌(PQQ)、苯醌和萘醌、N-氧化物、亚硝基化合物、羟胺、8-羟基喹啉、黄素类、吩嗪、吩噻嗪、靛酚和吲达胺。
术语“电极”定义为导电物质,其在电化学分析中保持稳定。电极材料的实例包括固体金属、金属糊、导电碳、导电碳糊和导电聚合物。
现在参考附图,在图1中阐明了生物传感器计量器,其总体由优选实施方案的参考字符100表示并且根据本发明的原理排列。生物传感器计量器100包括按照本发明的原理排列的生物传感器102。生物传感器计量器100包括微处理器104和相联的存储器106,所述存储器106用于存储程序和用户数据。将来自微处理器104的数字数据应用于数字-模拟(D/A)转换器108。D/A转换器108将数字数据转化成模拟信号。与D/A转换器108偶联的放大器110放大模拟信号。将放大器110的放大的模拟信号输出应用于本发明的生物传感器102。生物传感器102偶联放大器112。将放大的传感信号应用于模拟-数字(A/D)转换器114,其将放大的模拟信号转化成数字信号。将数字信号应用于微处理器104。
多数通过商业途径可获得的用于监控血液葡萄糖的一次性生物传感器需要沉积/印刷酶和介体与某种粘合剂的混合物。对于葡萄糖测量的应用,介体为氧化还原偶的氧化形式。取决于氧化还原偶,介体可以是非常强的氧化剂,如铁氰化物,从而与酶和粘合剂混合后化学地氧化官能团。随后,在墨水混合、储存和印刷过程中,少量还原的介体形成为试剂中的杂质。从而,混合和印刷试剂墨水的最终结果是产生还原形式的氧化还原偶,从而产生背景电流。该还原形式的介体的形成以及从而背景电流的形成可以随着不同批而异。该方法-产生的还原形式的介体,如从铁氰化物产生的亚铁氰化物通常可被氧化以用授予Genshaw等人并且转让给本受让人的美国专利号5,620,579和5,653,863中概述的算法使背景信号最小化。然而,该转换成校准截距的依赖方法的背景信号可以展开为值的范围。在这些发散的截距值的极限值,分析准确性通常将受到损失,因为不能分配合理的校准截距来适应该发散的截距。
根据本发明的特征,将在试剂中含有某一水平的还原形式的介体的一种等级的介体用于减小强氧化剂的作用。在热力学上,酶和介体的墨水混合物中少量还原形式的介体的存在可以减小从氧化形式向还原形式转化的驱动力。这通过向氧化的介体加入固定的少量的还原形式的介体来有利地实现。
即使将产生背景信号,但是美国专利号5,620,579和5,653,863中的算法将使背景的作用最小化来增加葡萄糖传感器的准确性。上面的专利公开了减小由于测量血液中特定分析物如葡萄糖所用的电流测定传感器中可氧化的杂质导致的背景偏倚的方法。如果传感器长时间段保存或者在应激(热、水分等等)下保存,那么由于传感器中还原介体增加的存在或者存在其他还原的杂质,如酶稳定剂,例如,谷氨酸,和具有还原当量的表面活性剂,这种传感器的背景电流将增加。例如,在基于铁氰化物的电流测定传感器中,背景偏倚与电极表面附近亚铁氰化物(从铁氰化物还原得来)的存在有关。与传感器使用中产生的亚铁氰化物(新鲜亚铁氰化物)相反,该积累的亚铁氰化物被氧化回铁氰化物以减小它导致的背景偏倚并从而延长了传感器保存期限。为了达到这个目的,所述方法使用电化学方法。当用算法纠正增进电化学方法时,进一步减小了背景偏倚。
所公开的方法涉及首先应用正电位脉冲(称作“熔化(burn-off)”脉冲),其在生物传感器的使用期间的正常电位谱之前。这通常通过在传感器的工作电极和参照电极之间施加0.1到0.9伏特(优选0.3到0.7伏特)的正电位1到15秒(优选5到10秒)来完成。熔化脉冲氧化最初的亚铁氰化物(或者其他可氧化的杂质),从而传感器可以以干净背景开始测定。通常,背景不是完全干净的,因为熔化脉冲仅氧化一部分可氧化的杂质。因为化学层覆盖工作电极和对电极,所以情况是这样的。在化学层中存在最初的亚铁氰化物,因为它来自铁氰化物。当加入样品流体并且化学层再水化时,工作电极附近的亚铁氰化物被再氧化。剩余的亚铁氰化物扩散到样品流体中并与葡萄糖混合。该部分最初亚铁氰化物的再氧化必定影响葡萄糖。流体测试样品应用后非常短的时间(几秒)内,最初亚铁氰化物位于电极附近。其原因是化学药品(酶和铁氰化物等等)作为薄层沉积在工作电极和对电极上。熔化技术利用了这点,因为可以熔化相当大量的最初亚铁氰化物而流体测试样品中分析物的浓度没有显著减小,大多数所述样品不直接与电极接触。实验已经证明正确地应用熔化脉冲可以将受到应激的传感器的背景偏倚减小40%。
将美国专利号5,620,579和5,653,863公开的方法有利地应用于使背景信号的作用最小化以增加优选实施方案的葡萄糖生物传感器计量器100的准确度。将上面专利的主题引入本文作为参考。
根据本发明的特点,额外量的还原形式的介体通过热力学性质使校准截距稳定同时维持校准斜率。根据在葡萄糖传感器中起作用的还原形式的介体(例如,亚铁氰化物)的功能,将它称作内部参照。
在1996年5月28日授予Bloczynski等人并且转让给本受让人的美国专利号5,520,786中描述了电化学活性有机分子介体的实例。具体地,包含本文中称作MLB-92的3-苯基亚氨基-3H-吩噻嗪的公开的介体(表1中的化合物18)已用于根据本发明的特征制造葡萄糖生物传感器102。将上面专利的主题引入本文作为参考。
通过商业途径可获得的生物传感器计量器和生物传感器由BayerCorporation以商品名Ascensia DEX生产和销售。Ascensia DEX生物传感器通常包括尽可能纯的形式的铁氰化物用作试剂。通过将足够量的亚铁氰化物加入纯的铁氰化物,Ascensia DEX生物传感器已经用于根据本发明的特征制造葡萄糖生物传感器102。将定义生物传感器102的内部参照的亚铁氰化物加入Ascensia DEX试剂墨水的益处包括增加截距而不改变斜率、使截距范围稳定和增加保存期间生物传感器的长期稳定性的直接好处。
根据本发明的特征,将具有较低氧化还原电位的MLB-92介体用于制造具有特定性质的葡萄糖生物传感器102。通过加入足量内部参照亚铁氰化物,新的生物传感器系统可以以两种工作电位工作:(1)在400mV下,其中新的介体和内部参照都被氧化,和(2)在100mV下,其中仅氧化新的介体。该方法的重要性是两重的。首先,这样制备的(新介体和内部参照)的葡萄糖生物传感器102可以在高电位(+400mV)下工作以在一定范围内产生电流,该范围满足现有仪器的硬件需要的校准特征。其次,因为介体的较低的氧化还原电位并且从而较低的氧化功率将几乎不能将介体的氧化形式转化成还原形式,所以可以对传感器应用较低工作电位(0-100mV)以便避免内部参照的氧化。从而,基于新的介体的一组新的校准特征由于较低的氧化功率最可能具有接近0的截距,所述特征将导致对于葡萄糖测量的更好的分析精密度。它还通过避免一些已知的可氧化的种类,如尿酸和对乙酰氨基酚的氧化而减小了全血中的基质干扰。
根据本发明的特征,内部参照对葡萄糖传感器102的另一应用是向生物传感器系统加入足够大量的内部参照以产生高电流响应。使用两步算法,其中在它们之间使用断路((Bayer专利#5,620,579和#5,653,863),第一个电位步骤设置在400mV以产生电流,其主要是由于内部参照信号,而第二步设置在低电位(0-100mV)以产生仅与葡萄糖浓度有关的电流信号。第一种信号(其几乎不依赖于全血血细胞比容)与低电位时的第二种信号的比可以用于校正由于血细胞比容影响导致的分析偏倚。
根据本发明的特征,将内部参照定义为可氧化的种类,其在一个实施方案中进一步定义为可逆氧化还原偶的还原形式,其具有与介体相等或者更高的氧化还原电位。内部参照的概念和使用在分析化学领域非常常见。然而,在现有专利或者文献中还没有提出对生物传感器使用内部参照的实例。在上述所有三种情形中,内部参照对于氧化两种种类的工作电位加性地增加响应电流,且对于分析物是葡萄糖的情况,总的响应电流通过如下表示:
I总的=I内部参照+I葡萄糖
I内部参照∝(内部参照)并且I葡萄糖∝(葡萄糖)
其中I内部参照是由于内部参照造成的总响应电流的部分,而I葡萄糖是由于介体氧化造成的,其与葡萄糖浓度成比例。
根据本发明的特征,内部参照可以是相同的介体种类或者具有比介体更高的氧化还原电位的可氧化的种类。从而,对于具有仅氧化介体的低工作电位的生物传感器,电流I内部参照将为0。然而,对于具有氧化两种种类的更高的工作电位的生物传感器,总响应电流将是由于内部参照导致的部分和由于葡萄糖导致的部分的总和。因为内部参照浓度是固定的,所以传感器的校准斜率将仅仅取决于葡萄糖的传感器响应而截距将取决于所加的内部参照的量。换句话说,内部参照将仅仅偏移截距并且不改变校准斜率。从而,内部参照的概念提供了制造葡萄糖生物传感器的新的且不同的方法。
现在参考图1B、1C和1D,根据用于本发明的葡萄糖生物传感器102的内部参照的使用,有至少3种工作模式。在恒电势下,三种工作模式在图1B、1C和1D中表示。所阐明的三种工作模式的每一种都包括第一个熔化脉冲,接着是第二个等待时间段或者断路,和最后第三个读数脉冲,每个脉冲或者时间段都具有所选的持续时间,例如,10秒。在基本和最直接的工作中,亚铁氰化物以总铁氰化物的0.1到1%的浓度保持在铁氰化物中,从而为本发明的葡萄糖生物传感器102提供了内部参照。这在图1B中描绘,其中第一和第三个时间段的电位都处于相同电压,例如,400mV。通过铁氰化物的合适的纯化方法或者通过向纯铁氰化物加入足量的亚铁氰化物,可以实现保持小百分比的定义内部参照的亚铁氰化物。这些保持方法的结果是在铁氰化物中有意保持所希望量的亚铁氰化物作为特定等级的铁氰化物。这与常规观念相反,常规观念是具有尽可能纯的形式的铁氰化物,如对于DEX试剂,通常以铁氰化物的0.05%或者更小的亚铁氰化物为杂质。DEX传感器的最终制剂中最希望的量是0.1%亚铁氰化物,其对于DEX传感器将导致将校准截距稳定在较窄的范围,同时维持校准斜率。
在图1C中,显示了第二种工作模式,其中向酶和具有比内部参照更小氧化还原电位的介体的试剂加入所需量的亚铁氰化物(内部参照)。对于现有仪器和具有新硬件需求的仪器,预期生物传感器102在高和低电位(例如,400mV和100mV对Ag/AgCl)下工作。该生物传感器可以在图1B中关于现有仪器100和图1C中关于新仪器100描述的电位程序中工作。介体和内部参照组合的实例包括MLB-92和亚铁氰化物以及钌六胺和亚铁氰化物的系统。两种氧化还原电位的分开足够大,从而当在低电压下工作时内部参照种类通常不氧化。
在图1D中,显示了第三种工作模式,其中向酶和具有低于内部参照的氧化还原电位的介体的试剂混合物加入较高但是所希望浓度的亚铁氰化物。内部参照的量将优选产生等于校准范围内全刻度的约50%到75%的电流。在工作算法中,将第一个电位步骤设置成氧化介体和内部参照两者(400mV),而读数脉冲的第二个电位步骤是仅氧化介体(0-100mV)。图1D的第一个电位步骤的电流将与紧靠着电极的内部参照最相关,并且应该几乎没有血细胞比容作用。来自第二个步骤与来自第一个步骤的电流的比对由于血细胞比容作用引起的分析偏倚提供校正。
已经进行了实验来显示向介体系统加入内部参照来克服现有问题或者增强根据本发明的生物传感器102的传感器性能的可行性。
现在参照图2A、2B和2C,显示了三个循环伏安图,阐明了本发明的生物传感器102的运行。所阐明的三个循环伏安图是关于0mg/dL葡萄糖的全血样品中基于MLB的葡萄糖生物传感器102的,其中以亚铁氰化物作为内部参照。
图2A阐明了工作电极对铁氰化物对电极的关系,图2B阐明了工作电极对银(Ag)和氯化银(AgCl)或者Ag/AgCl对电极的关系,且图2C阐明了工作电极对MLB-92对电极的关系。标记为1和2的各自峰分别代表对于所有三个伏安图,介体MLBred(MLB的还原形式)和内部参照亚铁氰化物的氧化。当对电极上的氧化还原偶从铁氰化物改变成Ag/AgCl到MLB-92时,MLBred的氧化峰沿着电位刻度偏移。然而,可以看到在图2A、2B和2C的所有三个伏安图中,介体MLB-92与内部参照亚铁氰化物的相对位置是相同的。
参考图3,图3是阐明了在不同电压工作电位下,本发明的生物传感器102的线性响应的图。生物传感器102在(1)400mV电位和(2)150mv电位下工作。图3阐明了以20mM亚铁氰化物作为内部参照的基于MLB-92介体的生物传感器102的线性剂量反应。标记为实例1和实例2的各自线来自对于Ag/AgCl对电极的400mV和150mV工作电位。如图3中所示,对于在400mV和150mV电位下工作,生物传感器102给出几乎相同的斜率但是不同的截距。该结果证明内部参照可以选择性被氧化或者通过工作电位而避免。从而,一个生物传感器102可以用于两个不同的计量器。
已经系统地制备了生物传感器102的实例,从而表明随着作为内部参照的亚铁氰化物增加,截距增加,而斜率保持几乎不变。在下面的制剂中制备了三种工作电极试剂。将这三种试剂针状沉积(pin-deposited)在两个形式的传感器上:(1)Ag/AgCl作为对电极,(2)10%印刷的铁氰化物作为对电极。
制剂 | 酶,PQQ-GDH | 介体MLB-92 | 内部参照铁氰化物 | 缓冲液和粘合剂 |
1 | 20单位/μL | 24mM | 0mM | 0.1M NaCl+磷酸盐,1%CMC |
2 | 20单位/μL | 24mM | 4mM | 0.1M NaCl+磷酸盐,1%CMC |
3 | 20单位/μL | 24mM | 8mM | 0.1M NaCl+磷酸盐,1%CMC |
图4阐明了使用本发明的生物传感器102,以10%印刷的铁氰化物作为对电极,所加入的内部参照对总体伏安电流的作用。图4提供了在0mg/L葡萄糖的全血样品中,以亚铁氰化物作为内部参照的传感器的循环伏安图。标记为A、B和C的伏安图分别使用制剂1、2和3,其都使用10%印刷的铁氰化物的对电极。
在图4中使用以10%印刷的铁氰化物作为对电极的传感器显示了所加入的内部参照对总体伏安电流的作用。这里主要的氧化/还原峰集中在约-0.38伏特对10%铁氰化物,这是由于介体MLB造成的。在约0-50mV的氧化峰是由于亚铁氰化物的内部参照造成的。在随着内部参照浓度从0增加到4到8mM,内部参照亚铁氰化物的氧化峰也增加时,介体的氧化峰几乎不变。这里MLBred的主要氧化峰不受内部参照存在的影响,这一事实进一步解释了内部参照的概念。
参考图5A和5B,其中显示了阐明以Ag/AgCl作为对电极的本发明的基于MLB的生物传感器102的线性响应和增加的内部参照导致的增加的截距的图。图5A阐明了使用分别标记为实例1、实例2和实例3的0、4和8mM亚铁氰化物,基于MLB的生物传感器102的线性剂量反应。图5B阐明了作为本发明的生物传感器102的工作电极试剂中加入的亚铁氰化物的函数的截距和斜率。所有三个传感器都使用Ag/AgCl作为对电极。
还参考图6A和6B,其显示了阐明以10%铁氰化物作为对电极的本发明的基于MLB的生物传感器102的线性响应和增加的内部参照导致的增加的截距的图。图6A阐明了使用分别标记为实例1、实例2和实例3的0、4和8mM亚铁氰化物,基于MLB的生物传感器102的线性剂量反应。图6B阐明了作为本发明的生物传感器102的工作电极试剂中加入的亚铁氰化物的函数的截距和斜率。所有三个传感器都使用10%印刷的铁氰化物作为对电极。
在剂量反应实验中,图5A和5B的使用Ag/AgCl对电极,和图6A和6B的10%铁氰化物对电极的传感器系列都显示线性响应和内部参照增加导致的增加的截距。为了实际目的,图5A和5B中的三个传感器的斜率不变而截距随着加入的亚铁氰化物线性增加。截距与所加入的亚铁氰化物的相同的线性关系和平坦斜率趋势在用%印刷的铁氰化物作为对电极的传感器系列中重复,如图6A和6B中显示。
已经进行了实验来显示向DEX试剂墨水加入亚铁氰化物,改变根据本发明的生物传感器102的校准截距而不改变斜率。
图7阐明了本发明的DEX型生物传感器102的校准截距与增加内部参照的线性关系。用在DEX传感器的标准DEX试剂中混合的0、0.02、0.04、0.06和0.08%亚铁氰化物制备图7中标记为BC7的固定形式中5种不同的制剂。在图7中显示了BC7形式的这5个传感器的回归斜率和截距。除了由于实验问题,使用0.06%亚铁氰化物的传感器之外,其他四个传感器的截距对于作为内部参照加入的亚铁氰化物的量都给出了良好的线性函数。另一方面,所有5个传感器的斜率都落入平坦线,表明加入内部参照不改变本发明的DEX型生物传感器102的斜率。
图8A和8B阐明了本发明的生物传感器102的来自对照试剂墨水和向20%铁氰化物的试剂混合物中加入0.1%亚铁氰化物的试剂墨水的残留亚铁氰化物的流动注射分析(FIA)产生的信号与参照比。向DEX试剂墨水加入内部参照亚铁氰化物的一个细微作用是减小介体铁氰化物向亚铁氰化物转化的驱动力。从而,铁氰化物成为DEX传感器中残余电流的来源。显示该细微作用的一种方法是在长时间段内监控使用内部参照的试剂墨水以及对照试剂墨水的残余电流(背景电流)的增加。两种试剂墨水都冷藏(2-8℃)保存几周。图8显示了来自两种试剂墨水的残留亚铁氰化物的FIA结果。从图8,信号与参照的比(S/R)代表与作为FIA中的参照加入的亚铁氰化物相比,来自试剂墨水的亚铁氰化物的相对量。从而,来自FIA分析的S/R值越高,试剂墨水中的亚铁氰化物越高。从图8A可以看出对于对照墨水和加入亚铁氰化物的试剂墨水两者,S/R值在6周的时间段内增加。然而,加入亚铁氰化物的试剂墨水曲线与对照曲线相比在6周的时间段内残余电流增加较缓慢。在图8B中,将来自对照墨水和加入亚铁氰化物的试剂墨水的S/R反应曲线合并用于比较。对于第一次近似值(因为两个二阶多项式的二次项的系数都非常小),对于在冷藏期间在6周内残余电流的增加率,加入亚铁氰化物的试剂墨水曲线比对照曲线小约30%([0.0918-0.0638]/0.0918=30%)。从而,从图8A和8B可以理解通过向根据本发明的生物传感器102的DEX试剂墨水加入内部参照亚铁氰化物,显著减小了试剂墨水中铁氰化物向亚铁氰化物的转化率。
尽管本发明已经参照附图中所示本发明的实施方案的细节进行了描述,但是这些细节不意在限制本发明的范围,本发明的范围由所附权利要求要求保护。
Claims (20)
1.用于测定测试样品中分析物浓度的生物传感器,其包含:
用于与分析物发生电化学反应的混合物;所述混合物包括酶,
介体,和
作为内部参照的可氧化的种类。
2.权利要求1的生物传感器,其中将所述内部参照定义为可逆氧化还原偶的还原形式,其具有与所述介体相等或者更高的氧化还原电位。
3.权利要求1的生物传感器,其中所述介体包含3-苯基亚氨基-3H-吩噻嗪。
4.权利要求3的生物传感器,其中所述内部参照包含亚铁氰化物。
5.权利要求4的生物传感器,其中定义所述内部参照的所述亚铁氰化物和所述介体在第一种电压电位下氧化,并且仅所述介体在第二种电压电位下氧化;所述第二种电压电位小于所述第一种电压电位。
6.权利要求5的生物传感器,其中所述第一种电压电位为约400mV,且所述第二种电压电位为约100mV。
7.权利要求1的生物传感器,其中所述介体包含铁氰化物。
8.权利要求7的生物传感器,其中所述内部参照包含亚铁氰化物。
9.权利要求1的生物传感器,其中所述介体包含钌六胺。
10.权利要求9的生物传感器,其中所述内部参照包含亚铁氰化物。
11.权利要求10的生物传感器,其中所述酶包含葡萄糖氧化酶。
12.包括酶、介体和作为内部参照的可氧化的种类的混合物的生物传感器的使用方法,所述方法包括步骤:
在第一个时间段应用第一种电压电位;
提供固定的延迟时间段;
所述延迟时间段后在最后时间段应用第二种电压电位;并且其中选择性提供所述第一种电压电位和所述第二种电压电位以仅氧化所述介体或者所述介体和所述内部参照两者。
13.权利要求12的方法,其中在第一个时间段应用第一种电压电位的步骤包括在第一个时间段应用所选的高的第一种电压电位以氧化所述介体和所述内部参照的步骤。
14.权利要求12的方法,其中在第一个时间段应用第一种电压电位的步骤包括在第一个时间段应用所选的低的第一种电压电位以仅氧化所述介体的步骤。
15.权利要求12的方法,其中所述延迟时间段后在最后时间段中应用第二种电压电位的步骤包括应用所选的第二种电压电位以氧化所述介体和所述内部参照的步骤。
16.权利要求12的方法,其中所述延迟时间段后在最后时间段中应用第二种电压电位的步骤包括应用所选的第二种电压电位以仅氧化所述介体的步骤。
17.权利要求12的方法,其中应用所述第一种电压电位和应用所述第二种电压电位的步骤包括应用100mV到400mV的所选的电压电位的步骤。
18.权利要求12的方法,其中应用所述第一种电压电位和应用所述第二种电压电位的步骤包括在第一个时间段应用所选的第一种电压电位以氧化所述介体和所述内部参照两者;和应用所选的第二种电压电位以仅氧化所述介体的步骤。
19.权利要求12的方法,其中所述生物传感器包括包含3-苯基亚氨基-3H-吩噻嗪和钌六胺之一的介体;并且其中内部参照包含亚铁氰化物;并且其中应用所述第一种电压电位和应用所述第二种电压电位的步骤包括应用所选的第一种和第二种电压电位以仅氧化所述介体的步骤。
20.权利要求12的方法,其中应用所述第一种电压电位和应用所述第二种电压电位的步骤包括应用所选的第一种和第二种电压电位以氧化所述介体和所述内部参照两者的步骤;其中所述内部参照在狭窄范围内有效地使校准截距稳定并且所述内部参照有效维持所述生物传感器的校准斜率。
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- 2005-02-04 CA CA002553632A patent/CA2553632A1/en not_active Abandoned
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- 2005-02-04 CN CNA2005800038597A patent/CN1914331A/zh active Pending
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- 2005-02-04 WO PCT/US2005/003622 patent/WO2005078118A1/en active Application Filing
- 2005-02-04 AU AU2005212396A patent/AU2005212396A1/en not_active Abandoned
- 2005-02-04 KR KR1020067015845A patent/KR20060131836A/ko not_active Application Discontinuation
- 2005-02-04 US US10/590,765 patent/US8696880B2/en active Active
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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CN110646479A (zh) * | 2019-06-27 | 2020-01-03 | 吉林化工学院 | 一种比率电化学传感器用于检测对乙酰氨基苯酚 |
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AU2005212396A1 (en) | 2005-08-25 |
US20070045126A1 (en) | 2007-03-01 |
US10067082B2 (en) | 2018-09-04 |
JP2007523326A (ja) | 2007-08-16 |
BRPI0507376A (pt) | 2007-07-10 |
CR8597A (es) | 2007-02-07 |
KR20060131836A (ko) | 2006-12-20 |
EP1713926A1 (en) | 2006-10-25 |
MA28350A1 (fr) | 2006-12-01 |
EP1713926B1 (en) | 2012-08-01 |
US9410917B2 (en) | 2016-08-09 |
US20160313272A1 (en) | 2016-10-27 |
CA2553632A1 (en) | 2005-08-25 |
US20140174951A1 (en) | 2014-06-26 |
US8696880B2 (en) | 2014-04-15 |
WO2005078118A1 (en) | 2005-08-25 |
NO20063939L (no) | 2006-11-03 |
RU2006132051A (ru) | 2008-03-20 |
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