CN1160563C - 用于确定试样检测初始时间的电化学分析 - Google Patents

用于确定试样检测初始时间的电化学分析 Download PDF

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CN1160563C
CN1160563C CNB001227203A CN00122720A CN1160563C CN 1160563 C CN1160563 C CN 1160563C CN B001227203 A CNB001227203 A CN B001227203A CN 00122720 A CN00122720 A CN 00122720A CN 1160563 C CN1160563 C CN 1160563C
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T��J���¹���
T·J·奥哈拉
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M·特奥多尔蔡克
M·Z·克尔马尼
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Abstract

一种电化学分析方法，包括：以高精度确定试样桥接电化学池电极之间缝隙的时间。该方法涉及向缝隙施加恒定的小电流，同时监测电极之间的电位差。试样开始检测后施加一恒电压，并且在一定的时间内监测流过试样的电流和/或电荷。由测得的电流或电荷，计算感兴趣的分析物浓度。

Description

用于确定试样检测初始时间的电化学分析
技术领域
本发明涉及一种用于测量生物液体中的分析物浓度的电化学设备；更具体地说，涉及确定该液体在设备的工作电极和参比电极之间产生电连接的时间的机理。
背景技术
涉及对生物液体进行测试的各种药物诊断方法，如：对血液，尿液，或者唾液进行测试，以便确定在该液体中分析物的浓度。在这些分析物中最具吸引力的是葡萄糖，并且含有酶基组合物的干相试剂条带广泛地用于临床实验，医师诊所，医院，和家庭中，以测试生物液体试样中的葡萄糖浓度。实际上，试剂条带已经成为许多国家的估计约1千6百万糖尿病人每日所必须的。由于糖尿病可导致血液化学危险的反常现象，它可能导致失明，肾功能衰竭，以及其它严重的医学后果。为了降低这些后果的危险性，许多忠有糖尿病的人必须定期进行自我测试，以便据此来调节他们的葡萄糖浓度，例如，通过控制日常饮食和/或通过注入胰岛素。一些患者必须每天测试他们血液中的葡萄糖浓度四次或更多次。
对于那些必须控制饮食以便控制摄入的糖量和/或控制胰岛素注入量的糖尿病患者，以及那些必须在频繁测试血液中葡萄糖浓度的指导下的患者而言，快速，廉价，并且准确的葡萄糖测量系统是特别重要的。
一种葡萄糖测量系统采用电化学方法，在干试剂条带上测试血糖的氧化作用。该试剂通常包括酶，例如：葡萄糖氧化酶或者葡萄糖脱氢酶，以及氧化还原介质，例如：二茂铁或者高铁氰化物。此种测量系统公开在Nakamura等人的US4224125(1980年9月23日出版)；Higgins等人的US4545382(1985年10月8日出版)；以及Nankai等人的US5266179(1993年11月30日出版)中，现在结合入本文以供参考。
电化学葡萄糖计的特征在于，根据该系统是否涉及分别测量电荷，电流或电压来决定采用电量计，电流计或电压计，从而确定葡萄糖浓度。在每种情况下，重要的是确定血液试样接触试剂的时间点，因为必须在此后的精确的时间期间向条带提供一电信号。
在Nankai等人的US5266179(1993年11月30日出版)中公开了一种测量血糖的电化学系统，其中试样应用时间被定义为在施以恒定电压的一对电极间产生电阻降的时间。
White等人的US5366609(1994年11月22日出版)中描述了与上述相同的原理，即监控电极之间的电阻降以确定向干葡萄糖试剂条带提供血液的时间。在上述两个专利中，向工作电极和参比电极间施以恒定电压，以追踪由于向干试剂条带提供血液试样而导致的电阻变化。
为了得到精确的结果，试样检测过程应在不扰乱分析物浓度的条件下进行，并且业已公开了许多种减少分析物微扰的技术。
Quade等人的德国专利DDR148387(1979年12月28日出版)中公开了一种电化学测量方法，使用一种新型的电子电路，可允许在恒电压(施加恒定电压)和恒电流(施加恒定电流)模式之间快速转换，同时还可减少电子元件的数量。该电路的目的是减少测量前试样的微扰。
Bartels等人的德国专利DDR208230(1981年11月2日出版)中公开了一种电化学测量方法，也试图减少试样的微扰。该测量设备包括一个电路，该电路中用二极管减少测量前的电流，而不使用额外的电流控制环路。而且，该电路以精确、快速的方式转换为电压模式。
Littlejohn等人的US4940945(1990年7月10日出版)中公开了一种可以测量血液试样pH值的便携式设备。该设备检测室中试样的方法是向位于试样室外的填充电极和位于试样室内的两个电极之一通入恒电流。当阻抗下降至少两个数量级时，该测量计识别出已经提供了足够的试样并发出嘟嘟声。而后，切断填充电极的电路，该电路中包括试样室内的两个电极，并且进行电压测量。
发明内容
本发明涉及一种测量在生物液体中的分析物浓度的方法，该生物液体提供给电化学诊断条带，该类型的条带包括并列设置的工作电极和参比电极，该方法包括：
(a)在工作电极和参比电极之间施加一预定的恒电流源，
(b)监控电极之间的电位差，
(c)将试样提供给条带，
(d)当电位差低于预定的阈值电压时进行记录，以确定试样检测时间，
(e)向试样施加一预定的恒电压，
(f)使条带与电流源断开，
(g)在施加恒电压后，在预定的时间测量电响应，以及
(h)用测得的电响应计算分析物浓度。
本发明提供了一种测量生物液体试样中分析物浓度的方法，该试样提供给电化学诊断条带，该类条带包括并列设置的工作电极和参比电极。该方法包括：
(a)在工作电极和参比电极之间施加一预定的恒电流源，
(b)监控电极之间的电位差，
(c)将试样提供给条带，
(d)当电位差下降至低于预定的阈值电压时进行记录，以确定试样检测时间，
(e)向试样施加一预定的恒电压，
(f)在施加恒电压后，在预定的时间测量电响应，
(g)用测得的电响应计算分析物浓度。
测量提供给诊断条带的生物液体试样中分析物浓度的测量汁，包括，电连接的下述装置：
(a)向工作电极和参比电极之间施加预定电流的装置，
(b)监控电极之间电位差的装置，
(c)用于确定电位差低于指示试样检测的预定阈值电压的装置，
(d)对试样检测作出响应，以便向试样施加预定的恒电压的装置，
(e)测量最终电响应的装置，以及
(f)通过使用测得的电响应计算分析物浓度的装置。
本发明提供一种用于测量分析物浓度的电化学方法和设备，包括以高精度确定将试样提供给电化学诊断条带的反应区并桥接电极之间缝隙的时间。精确地确定试样施加时间(更具体地说，试样检测时间；我们使用可替代的术语)允许对试样进行更精确和更高精度的分析。
本发明确定试样使用时间的方法，其优点在于，与施加恒电压的现有技术相比，本发明施加恒定的小电流来检测试样减少试样的微扰。使用后一种方式，施加试样时将导致电流超过限定的阈值，则计时开始。由于取样速率受限，该电流一般相当大，在传感器感知以前就已经超过了阈值。当观察到大电流时，观察到介质中有相当大的微扰。这将导致不精确测量，特别是对于低分析物浓度。
现有技术施加恒电压以检测施加试样的方法还有另一个缺点，即初始电流一般随着分析物浓度的减少而下降。因此，更难以确定低分析物试样的初始试样检测时间。根据相同的原因，如果电流阈值设定得太低，由于干扰可能导致错误地触发。由于进一步的复杂原因，存在高浓度的血红细胞也可导致初始电流的下降。
分析物浓度和血红细胞浓度不影响本发明的方法。同样地，干扰也不是主要问题，因为检测的触发器是电压信号的大幅变化。
附图说明
图1描绘的是本发明试样检测方法中施加的电流—时间和测得的电压—时间的图。
图2是本发明分析方法中施加的电压—时间和最终电流响应—时间的图。
图3是本发明可替代的分析方法中施加的电压—时间和电流响应—时间的图。
图4是本发明另一个可替代分析方法中电荷—时间的图。
图5描绘的是适合用在本发明分析方法中的电化学设备图。
图6是适合用于本发明的电路图。
具体实施方式
本发明涉及一种测量生物液体中分析物浓度的电化学方法。为了简单起见，下面将重点描述测量全部血液试样中的葡萄糖浓度；然而，本领域中的熟练人员能够了解如何将该方法用于检测在其它液体(如：唾液，尿液，间质液体等)中的其它分析物(如：胆固醇，酮体，酒精等)。
用于测量液体试样中分析物浓度的电化学(电流)方法包括将试样置于电化学池的反应区中，该电化学池具有两个电极，其阻抗适合于进行电流测量。该分析物可直接与电极反应或与氧化还原剂反应生成可氧化(可还原)的物质，其生成量相应于分析物的浓度。该可氧化(可还原)物质的含量用电化学方法确定。此类分析方法必须精确限定试样在反应区进行测试的时间点。这可允许在提供试样后立刻施加电化学波形(即电压)以及精确限定潜伏期或反应时间。相反，这可以提高分析的精确度和精密性，如下文所述。
本发明提供一种用于确定试样检测时间的改进方法和设备。该方法包括向电化学诊断条带的电极提供小的恒电流源，并监测电极之间的电位差。由于在电极之间存在一于燥缝隙，因此开始时流过一可忽略的电流。当试样提供给该条带并填充该缝隙时，测量电压快速下降，导致测试时间开始。以这种方式确认已经提供了试样，并且该设备由恒电流模式转换为恒电压模式。在恒电压模式中，测量作为时间的函数的电流或电荷，以便计算分析物浓度。该项技术减少了由于时间—初始电路造成的信号响应误差，并因此允许低的检测限制。电子组件简单且成本低。
图1描绘的是本发明试样检测方法中施加的电流和测得的电压的图。在零时间之前(即在试样引入前)，在电极之间施加恒电流(例如此处为1μA)，但仅仅是可忽略的电流流动。更小的电流可减少微扰，并且优选地特别适用于低的分析物浓度。测得的电压由电路的电源电压决定——此处为5V。当将试样引入池中时(在零时间点)，施加的电流可以在两个电极之间流动，并且测得的电压快速下降。当该电压降至低于阈值电压时，该设备从施加恒电流转换为施加恒电压。
图2描绘的是试样检测后，作为时间的函数的施加电压与测得电流的图。试样在时间t＝0开始进行检测，其后立刻向工作电极和反电极之间施加电压。其结果是，在电极之间有电流流动。一旦体系建立起来，使用已知分析物浓度的试样，在预定的时间后该电流作为分析物浓度的量度。预定时间的长短没有严格限制。当液体为血液而分析物为葡萄糖时，一般至少为约3秒。该期间一般可提供足够的时间以便使试剂溶解，并且减少易于测量的介质的用量。在高血细胞比容的情况下，所有物质都是相同的，需要较长的时间溶解。实际上，一般使用者感兴趣的是越快越好地读数。10秒的时间一般令人满意，不必等更长的时间。当然，一旦确定了预定的时间，精确和精密的结果需要每次均使用相同的时间。在任何情况下，精确的电流确定依赖于精确的t＝0的确定。
图3描绘的是在可替代方法中，测得电流和施加电压相对于时间的图。在该方法中，第二个电压脉冲在预定的时间之后施加给电极。一般地，该第二个脉冲在预定时间之后立即施加(以便减少总测量时间)，但也可允许延迟。再次，具有再现性的结果需要具有再现性的过程；因此，在该方法中，精确确定t＝0点也是重要的。该第二个脉冲导致流过电极的电流中产生正峰，随后是衰减的电流。在体系建立起来后，该分析物浓度可以单独由衰减速率确定，也可与图2中描绘的电流测量共同确定。一般地，在该第二个脉冲后，该电流呈周期性的指数衰减，开始为约1秒随后持续至少几秒钟。
图4描绘的是图3的方法，其中测量的是电荷而不是电流。根据图3，分析物浓度可以从在固定时间的总电荷和/或从施加该第二个电压之后的衰减速率来确定。
图5描绘的是适用于下述方法的“薄层”设备10。基片12为其上沉积有Pd涂层16的聚酯基体14，从而形成工作电极，沉积方法一般为溅射。由缓冲剂、介质和酶组成的干试剂沉积在电极一端18附近。间隔层20是具有限定出电化学池的剪切块22的双侧粘合层。一般地，该间隔器的厚度小于约200μm。顶层24为其上沉积有Au层28的聚酯层26，从而形成参比电极，沉积方法一般也为溅射。
上述类型的设备可以用葡萄糖氧化酶(GOD)/高铁氰化物体系，通过下述反应来确定葡萄糖浓度，其中GOD*为还原的酶。
反应1
葡萄糖+GOD→葡萄糖酸+GOD*
反应2
GOD*+2高铁氰化物→GOD+2亚铁氰化物。
高铁氰化物([Fe(CN)6]3-)作为介质，将GOD*还原为其催化态。只要还存在过量的介质，GOD和酶催化剂就将继续氧化葡萄糖。亚铁氰化物([Fe(CN)6]4-)是总反应的产物。理想地，开始时没有亚铁氰化物，但是实际上往往存在少量的亚铁氰化物。该反应完成后，亚铁氰化物的浓度(用电化学法测量)表明葡萄糖的初始浓度。总反应是反应1与反应2的和。
反应3
                  GOD
葡萄糖+2高铁氰化物→葡萄糖酸+2亚铁氰化物
“葡萄糖”特指β-D-葡萄糖。
在PCT申请WO97/18465中详细描述了该体系，在此处结合入本文以供参考。
图6描绘的是适合用于本发明的电路的实施方案。开始，在位置1通过开关105向该条带施以恒电流源。该恒电流源由操作放大器104，电压基准102，以及电阻器101和103组成。该电流由电压基准102和电阻器103的比值确定。电阻器101用于产生所需的偏流。操作放大器110和电阻器109用作电流—电压转换器。开始，在条带上没有试样，点107和108之间的电阻非常大，并且流过条带的电流可以忽略。在此条件下，操作放大器104输出的电压(V1)高。当将试样提供给条带时，其电阻显著下降，并且由于恒电流流过条带，V1下降。V1通过模拟—数字转换器111输入到微处理器112。微处理器112确认该电压降作为试样检测的开始，开关105置于位置2以便使条带与电流源断开，并使其与电压源106连接。在此种情况下，计时电流测量可以通过测量操作放大器110的输出电压(V2)获得。该电压与流过条带的电流成比例。
下面的实施例用于说明本发明，但是并不作为对本发明的任何限制。
                           实施例
建立图6中的电路，其中条带S为如图5所示类型的薄层电化学葡萄糖条带，并具有Pd和Au电极。该Pd电极用缓冲剂，葡萄糖脱氢酶(PQQ)以及高铁氰化物涂覆。向该干葡萄糖条带的工作电极和反/参比电极之间施加恒定的、小(-1μA)而无干扰的电流。由于该条带是干燥的，工作电极和反/参比电极之间的电阻基本上是无限大。当全部血液试样均施加给该池后，观察到电压下降。阈值为约50～500mV时为开始时间(优选的是阈值为约300mV)。当试样检测后，该设备由施加恒电流转换为施加恒电压。由该试样测得作为时间函数的电流值，从而计算葡萄糖浓度。
本领域的熟练人员应能理解，上述描述和实施例用于说明本发明，但是并不限制本发明。可以对现有的描述进行改进，而不超出本发明的精神和范围。

Claims (12)

1、一种测量在生物液体中的分析物浓度的方法，该生物液体提供给电化学诊断条带，该类型的条带包括并列设置的工作电极和参比电极，该方法包括：
(a)在工作电极和参比电极之间施加一预定的恒电流源，
(b)监控电极之间的电位差，
(c)将试样提供给条带，
(d)当电位差低于预定的阈值电压时进行记录，以确定试样检测时间，
(e)向试样施加一预定的恒电压，
(f)使条带与电流源断开，
(g)在施加恒电压后，在预定的时间测量电响应，以及
(h)用测得的电响应计算分析物浓度。
2、根据权利要求1的方法，其中测得的该电响应为在预定的时间流过该试样的电流。
3、根据权利要求1的方法，其中测得的该电响应为从试样检测时间开始到预定时间流过该试样的电荷。
4、根据权利要求1的方法，还包括：在预定时间后施加第二预定电压，并且在施加该第二预定电压后测量第二电响应。
5、根据权利要求4的方法，其中该第二电响应为流过试样的电流的衰减速率。
6、根据权利要求4的方法，其中该第二电响应为施加第二电压后，在预定衰减期间，流过试样的电荷。
7、一种测量在生物液体试样中的分析物浓度的测量计，该生物液体被提供给诊断条带的工作电极和参比电极之间，该测量计包括电连接的下述装置：
(a)向工作电极和参比电极之间施加预定电流的装置，
(b)监控电极之间电位差的装置，
(c)用于检测电位差低于指示试样检测的预定阈值电压的装置，
(d)对试样检测作出响应，以便向该试样施加预定的恒电压的装置，
(e)测量最终电响应的装置，以及
(f)通过使用测得的电响应计算分析物浓度的装置。
8、根据权利要求7的测量计，其中测量最终电响应的装置为安培计。
9、根据权利要求7的测量计，其中测量最终电响应的装置为库仑计。
10、根据权利要求7的测量计，还包括：向该试样施加第二预定电压的装置，以及测量第二最终电响应的装置。
11、根据权利要求10的测量计，其中测量第二最终电响应的装置为安培计。
12、根据权利要求10的测量计，其中测量第二最终电响应的装置为库仑计。
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