CN1143132C - 可用肉眼观察的血糖检测条 - Google Patents
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Abstract
可用肉眼观察的血糖检测条含有两个分别掺入了试剂的膜,所述试剂与施加到膜上的血样中的葡萄糖反应引起颜色改变。其中一个膜还含有抑制剂和染料。施加到条上的血样使两个膜形成两种不同的颜色。将颜色与校正颜色表进行比较,可使使用者确定血样中的葡萄糖浓度。
Description
技术领域
本发明涉及测量血糖浓度的干试剂条;更具体地讲,涉及无需测量仪即可进行葡萄糖测量的条。
背景技术
为了测量生物学液体中特定分析物的浓度,已经开发了许多可用肉眼观察的检测装置。这些装置例如可测量血液、尿或唾液中的葡萄糖、胆固醇、蛋白质、酮、苯丙氨酸或酶。
目前最常使用的装置是血糖测量仪。仅在美国,估计有一千四百万人患有糖尿病。为了避免严重的医学后果如视力丧失、循环问题、肾衰竭等,许多患糖尿病的人有规律地监测其血糖,然后采取必要的措施使其葡萄糖浓度保持在可接受的范围内。
这些装置中所用的试剂条含有一种指示剂,根据应用于所述条的全血样品中的葡萄糖浓度,所述指示剂可转变成不同的颜色。尽管一些试剂条利用还原性化合物,但是更常见的是,他们含有一种可氧化染料或染料对。一些试剂条含有酶,例如葡萄糖氧化酶,这种酶可将葡萄糖氧化成葡萄糖酸和过氧化氢。试剂条还含有可氧化染料和具有过氧化活性的物质,所述物质能够在存在过氧化氢的条件下选择性地催化可氧化染料的氧化。
美国专利3298789(1967年1月17日公布,R.L.Mast)公开了一种掺入了试剂组合物的吸水纸条,当将含葡萄糖的血液加至其表面时,所述吸水纸条的颜色发生变化。在使用时,将血液加至纸条表面一分钟后,擦拭血液,然后将纸条的颜色与颜色表进行比较,颜色表中有代表具体葡萄糖水平的颜色方块。
美国专利3630957(1971年12月28日公布,H.Rey等)公开了一种用试剂组合物涂覆的塑料箔条。使用方法与早期专利中所公开的类似—加血液,等约一分钟,将所得的颜色与颜色表进行比较。
美国专利4975367(1990年12月4日公布,Albarella等)公开了一种颜色匹配条,通过利用两种不同的指示剂系统,所述条在不同的分析物浓度产生不同的色度。该专利还公开了包括用于延缓不同系统的催化作用的颜色阻滞剂,改变特定浓度的分析物所产生的最终色度的方案。
美国专利5200325(1993年4月6日公布,J.M.Blatt等)公开了一种“自显示”条,其中不需进行颜色表比较。通过在指示剂反应前完成消去反应,所述条可表明是否存在预定浓度的分析物。因此,只有当存在预定浓度或更高浓度的分析物时,所述条才会产生预定的反应水平。
上述专利是“擦拭”条,其中通过擦拭所述条表面的过量血样而使全血颜色和指示剂染料所显示的颜色之间的干扰降至最小。其他方法包括将血样加至所述条的一个表面,然后在背面测量颜色。下述专利即是例子。
美国专利4935346(1990年6月19日公布,R.Phillips等)公开了一种测量仪、条以及用于确定全血样品中的葡萄糖浓度的方法。该方法包括简单地将全血样品加至用试剂浸渗的惰性多孔基质的第一(“样品”)表面。所述样品向背面“检测”表面迁移,葡萄糖与试剂反应产生吸收光的反应产物。检测表面的反射比读数可表明葡萄糖浓度。在两个不同的波长完成反射比测量以消除干扰。用通过基质的样品润湿的检测表面而引起的反射比的开始降低来引发定时回路。
美国专利5306623(1994年4月26日公布,Kiser等)公开了一种可用肉眼观察的血糖检测条,其中在分离出血红细胞并将样品与条上的试剂反应后,将含葡萄糖的全血样品加至条的一侧,然后在另一侧读出葡萄糖的读数。不对称的聚砜膜可用作这种条的单层基质。(参见美国专利5418142,1995年5月23日公布和美国专利5719034,1998年2月17日公布)。
美国专利5563031(1996年10月8日公布,Y.S.Yu)公开了用于检测生物学液体中的分析物如葡萄糖的干试剂条的染料对。所述染料对含有间[3-甲基-2-苯并噻唑啉酮腙]N-磺酰基苯磺酸一钠与8-苯氨基-1-萘磺酸铵(MBTHSB-ANS)并在产生强氧化剂如过氧化氢的级联反应中用作指示剂。所述染料对的优点是可溶于水溶液,但氧化偶联后不溶于水,由此降低了褪色并提供了稳定的终点。
White-Stevens和Stover,Clin.Chem.28,589-595(1982)讨论了由抗坏血酸引起的对诊断检测的干扰。抗坏血酸在利用过氧化物酶和氧化还原作用指示剂的检测物的颜色显色中可引起时间滞后。
M.J.Sherwood等Clin.Chem.29,438-445(1983)讨论了一种与校正的颜色级结合使用的可用肉眼观察的试剂条。所述条使用两个垫,每个垫包含正常范围的不同部分。该技术成为VisidexTM试剂条的基础,该试剂条可用肉眼观察。其他市售的可用肉眼观察的条包括DextrostixTM试剂条,Chem-strip bG和SmartStripTM试剂条。
发明内容
根据本发明,可用肉眼观察的血糖检测条包括:
a)供在主要表面接受血样并使样品穿透到次主要表面即背面的铺展层;
b)含第一和第二个膜的中间层,这两层膜基本上是并行的,每个膜均含有与铺展层的次主要表面相连的上主要表面以接受部分血样,并且每个膜均含有可与样品中的葡萄糖反应的试剂,当葡萄糖穿过膜时可引起试剂的颜色变化,第二个膜还含有抑制剂和染料,和
c)支持其他层并透过膜可观察膜上的任何颜色变化的支持层。
在本说明书和权利要求书中所用的“试剂”指与仅在第二个膜上或膜中的抑制剂和染料相对的,可在两个膜上或膜中的组分。所述染料是惰性的,即其颜色与葡萄糖浓度基本上无关。其目的是提高两个膜间的颜色差异。
本发明检测条的优点是无需擦拭步骤,可使用少量血样并可迅速得到结果。
附图说明
图1是本发明检测条的俯视图。
图2是本发明检测条的仰视图。
图3是本发明检测条另一实施方案的部件分解透视图。
图4是本发明检测条另一实施方案的部件分解透视图。
图5是用本发明实施方案测量的葡萄糖值对在标准测量仪上测量值作出的散布图。
本发明提供了一种不用擦拭的血糖条,所述条可用少量血样并且不需要测量仪就可快速确定血糖。图1是本发明的局部剖示俯视图,图2是仰视图。其中条10有三个部分:铺展顶层12,含试剂的中间层14。支持层16。中间层14由两个并行的膜14a和14b组成,这两个膜分别透过支持物16上的开口16a和16b可看到。膜14a含有与葡萄糖反应产生可用肉眼观察的颜色变化的试剂。膜14b也含有与葡萄糖反应引起颜色改变的试剂,但是所形成的颜色与在膜14a中形成的不同(下文讨论)。
在检测过程中,只需10μL以下的血样,将所述血样加在铺展顶层12上。由于样品透过层12,铺展开,因此,样品基本上均匀地分布到膜14a和14b上。血样中的葡萄糖随其穿透至支持层16上而与膜中的试剂反应产生颜色。如果支持层16不透明,可透过开口16a和16b观察颜色并将颜色与校正颜色表进行比较以确定血糖浓度。
许多材料都适用于作为铺展层;例如,纸、玻璃纤维、聚合物纤维、烧结塑料、织造或非织造织物以及膜。优选的材料只需很少样品,能够快速吸收样品,将其均匀分散至膜上并且不会影响膜中的试剂的化学性。当然,成本也是一个实际的问题。一种优选的铺展层材料是可从Tetko,Inc.,Deprew,NY买到的亲水聚酯织造网。所需的样品量很少和快速吸收说明是一种薄层。但是,与膜的良好接触非常重要,然而,如果该层很薄以致很容易皱就很难与膜有良好的接触。市售的Tetko网7-280/44是市售网中较厚的,有255微米,并且不容易皱。Tetko网吸收迅速并且有低的最小样品量;但是可能不能吸收大量血样,在铺展层表面(不理想地)剩下过量样品。
另一种优选的铺展层材料是热烧结塑料,如聚乙烯或聚丙烯,所述材料通过用血相容性表面活性剂预处理或后处理而具有亲水性。一种所述的材料是甲基油酰牛磺酸钠处理的多孔聚乙烯并可从Fairburn,Georgia的Porex Corp.买到。这种材料的优点是具有不寻常的强吸收作用,它可使液体离开表面,否则将会转移至所接触的物体或人。所述材料的孔为约20微米至约350微米,优选50至约150微米,或者平均约100微米。约0.5-0.6mm厚,约5-6mm宽,约30mm长的网是适宜的。
图3是可接受较大量样品的检测条的分解透视图。所述的条除具有图1和2中所示的构成外,还含有一任选的覆盖层18,在该层上有孔20供施加样品至铺展层。
图4是本发明另一种条的分解透视图。在该实施方案中,还有任选的吸收层22与铺展层12相邻以吸收过量样品。当铺展层12是较厚的烧结聚乙烯(Porex)时,通常不需要吸收层22。但是当铺展层是织造网(Tetko)时,由于所述网包含液体的能力较小,任选有吸收层22。
任选地,将所述条的部件用粘合剂粘在一起,双面胶是一种很方便的粘合剂。图4的实施方案包括粘合层24(带有开口24a和24b),用于将支持层16与中间层14相粘;粘合层26用于将中间层14与铺展层12相粘;以及粘合层28(带有开口28a)用于将覆盖层18与铺展层12和吸收层22相粘。粘合层26显示与膜14a和14b邻接;但是它们相对位置并不重要。因此,在膜和粘合层之间可有一缝隙或者它们可略有重叠。
最适用于本发明条的膜材料和试剂可提供快速反应、使用最少量的样品并产生均匀的颜色和稳定的终点。另外,血样颜色应基本上被不暴露于观察侧,从而不会干扰评估。基于这些标准,优选的膜材料是不对称的聚砜膜,该膜的孔径从一面至背面逐渐增加。(参见美国专利4629563。1986年12月16日公布,Wrasidlo)。这种膜可从美国Filter Co.San Diego,CA的Memtec Div.买到。这些膜在不到45秒钟的时间内,用10微升或更少样品可产生均匀的颜色显色和稳定的终点。所述膜避免血样颜色暴露于观察侧的能力取决于其孔径大小。大孔径不能很好地掩蔽血样颜色,但是孔径小于约0.10微米的膜在用试剂涂覆后有可能断裂。对不对称膜设计的孔径较小。优选使用孔径小于约1.0微米的膜,更优选约0.15-0.35微米的膜,所述膜在血细胞比容为30-55%时具有可接受的血样掩蔽效果。所述膜包括Memtec膜BTS30、45、55和65。优选膜14a和14b是相同的材料。通常需要约10微升血样来饱和检测垫,但是可以吸收达到由铺展层材料的孔隙率和尺寸所决定之体积的过量血样。
掺入到两个膜中的试剂组合物与血样中的葡萄糖反应,引起可检测的颜色变化。适宜的组合物是在上述专利4935346和5563031中公开的那些,这些专利引入本文作为参考。通常,这些组合物含有用于将葡萄糖转变成过氧化氢的组分,如葡萄糖氧化酶,和一种或多种用于检测从样品中的葡萄糖产生的过氧化氢的组分。用于检测过氧化氢的组分可以是过氧化物酶,优选辣根过氧化物酶,以及在反应过程中改变颜色的“指示剂”。所述指示剂可以是可氧化染料或染料对。过氧化物酶在存在过氧化氢的条件下催化所述指示剂的氧化。所述指示剂可以是3-甲基-2-苯并噻唑啉酮腙盐酸盐结合3,3-二甲基氨基苯甲酸(MBTH-DMAB)或8-苯氨基-1-萘磺酸铵(MBTH-ANS)。或者,指示剂可以是间[3-甲基-2-苯并噻唑啉酮腙]N-磺酰基苯磺酸一钠结合8-苯氨基-1-萘磺酸铵(MBTHSB-ANS)。后一种组合是优选的。
膜14b还可包含延缓颜色形成反应的抑制剂以及染料。膜14a仅含有颜色形成试剂组合物,可区别数种低葡萄糖水平;如25、50和80mg/dL。对于较高葡萄糖浓度,颜色太暗而无法可靠地区别葡萄糖水平。抑制在膜14b中的颜色显色,根据所用的抑制剂浓度,以便在较高葡萄糖水平时才有颜色显色。许多不同的抑制剂均是适宜的,包括2,3,4-三羟基苯甲酸,没食子酸丙酯和抗坏血酸(参见上述美国专利5306623)。抗坏血酸是优选的,优选选择抗坏血酸的浓度以便在葡萄糖水平达到约120mg/dL时才有颜色显色。
包括惰性染料与抑制剂产生了不同的颜色以便更好地区别葡萄糖水平。适宜的惰性染料是媒染黄(mordant yellow)、亮黄苯胺染料、双磷酸伯氨喹、噻唑黄G、亮黄和无水碱性嫩黄0(auramine0-anhydrous)。改变染料类型和浓度,可产生绿、黄、紫或蓝等颜色。0.1%的无水碱性嫩黄0是优选的,因为与MBTHSB-ANS结合可产生亮绿色,在葡萄糖水平之间有最大肉眼分辨率,并具有优秀的涂覆均匀性。在组合中,在膜14a和14b中的优选试剂组合物可使使用者区别从25到600mg/dL范围内的8个葡萄糖浓度水平。
为了进一步提高血样掩蔽效果,并由此可测量更宽范围的样品血细胞比容,可将各种水溶性带电聚合物加到试剂中,包括聚乙烯吡咯烷酮(PVP)、GAFQUAT*,聚丙烯酸和聚丙乙烯磺酸。根据经验,PVP是优选的。
具体实施方式
下列实施例以本发明的各种实施方案说明本发明,但不以任何方式构成限制。
实施例1
用孔径为0.2微米(Memtec BTS-55)的亲水不对称聚砜膜制备试剂条。通过使钝侧(较大孔径)与溶液1接触来涂覆膜(挂胶)。用玻璃棒擦拭膜条除去过量溶液。在56℃强迫空气箱中将膜进行空气干燥10分钟。
涂覆组合物是:
溶液1
水 25 mL
柠檬酸 0.282 g
柠檬酸三钠 0.348 g
甘露醇 0.25 g
EDTA 0.021 g
Gantrez 0.1125 g
Crotein 0.36 g
葡萄糖氧化酶 0.36 g(125单位/mg)
辣根过氧化物 0.071 g(501单位/mg)
Carbopol溶液* 1.25 mL
*(在1.25mL MeCN中含0.1375g)
柠檬酸缓冲液* 3.75 mL
*(在5.0mL水中含0.13g柠檬酸二钠)
对于低范围膜(14a),然后将溶液1涂覆的膜用染料溶液2挂胶,然后按上述在相同的箱中干燥5分钟。
染料溶液2组成是:
溶液2
溶剂* 38.16 mL
MBTHSB 0.1552 g
ANS 0.2142 g
20%Maphos(溶剂) 1.84 g
(*溶剂:50%水,30%EtOH和20%MeOH)
对于高范围膜(14b),用溶液1涂覆的膜然后用染料溶液3挂胶,然后按上述在相同的箱中干燥5分钟。
染料溶液3的组成是:
溶液3
溶剂* 38.16 mL
MBTHSB 0.1552 g
ANS 0.2142 g
20%Maphos(溶剂) 1.84 g
碱性嫩黄0 0.0316 g
抗坏血酸 0.13 g
(*溶剂:50%水,30%EtOH和20%MeOH)
将所得的膜切成0.6cm宽的条,然后制成图1和2所述的条。
通过用肉眼与颜色表(表1)比较,检测所述条,所述颜色表构成Munsell颜色芯片并校正到0-600mg/dL的葡萄糖范围。对288份通过尖钉取的静脉血样进行的随机检测与在标准血糖测量仪--YellowSprings Instruments(YSI)2300型STAT葡萄糖分析仪上测得的结果—对血浆葡萄糖值校正过的值有极好的相关性(R2=0.965)(图5)。
实施例2
重复实施例1的方法,不同的是将溶液1和2(或3)涂覆到膜的小孔侧,将掩蔽血样的聚合物涂层加到染料溶液涂层上。实验了许多不同的聚合物。下列各种水溶液在掩蔽血样颜色中是有效的:
聚乙烯吡咯烷酮-MW160000,2%GAFQUAT 734(季化的乙烯吡咯烷酮和甲基丙烯酸二甲基氨基乙基酯的共聚物)-MW 60000-100000,7%
聚苯乙烯磺酸的钠盐-MW 70000,7%
表1颜色表的Munsell Notation*
葡萄糖水平(mg/dL)0255080120180240400600 | 垫#1 垫#210 P 7/2 10 YR 7/65 PB 6/2 2.5 Y 6/810 B 6/4 2.5 Y 6/810 B 5/4 2.5 Y 6/82.5 PB 5/6 7.5 Y 6/62.5 PB 4/8 5 GY 5/65 PB 4/8 10 GY 5/67.5 PB 3/10 5 G 4/65 PB 3/8 10 G 3/4 |
*参见The Munsell Book of Color,Macbeth Div.ofKollmorgen Instruments Corp.,New Windsor,NY.
Claims (10)
1.可用肉眼观察的血糖检测条,包括
a)供在主要表面接受血样并使样品穿透到次主要表面即背面的铺展层(12);
b)含第一和第二个膜的中间层(14),这两层膜是并行的,每个膜均含有与铺展层的次主要表面相连的上主要表面以接受部分血样,每个膜均含有可与样品中的葡萄糖反应的试剂,当葡萄糖穿过膜时可引起试剂的颜色变化,第二个膜还含有抑制剂和染料,和
c)支持其他层并透过膜可观察膜上的任何颜色变化的支持层(16)。
2.权利要求1的条,其中所述铺展层包含烧结的聚乙烯。
3.权利要求1的条,其中中间层的每个膜均是不对称聚砜膜。
4.权利要求1的条,其中在每个膜中的试剂相同。
5.权利要求4的条,其中所述试剂包括间[3-甲基-2-苯并噻唑啉酮腙]N-磺酰基苯磺酸一钠结合8-苯氨基-1-萘磺酸铵。
6.权利要求4的条,其中所述试剂还含有可溶性聚合物。
7.权利要求1的条,其中所述抑制剂包括抗坏血酸。
8.权利要求1的条,其中所述染料包括无水碱性嫩黄0。
9.权利要求1的条,其中还包括在铺展层上的覆盖层(18),该层含有可施加样品于铺展层上的通过孔。
10.权利要求1的条,其中还包括与铺展层相连的吸收层(22)以吸收过量样品。
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US09/133,857 US6162397A (en) | 1998-08-13 | 1998-08-13 | Visual blood glucose test strip |
US09/133857 | 1998-08-13 |
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CN1247314A CN1247314A (zh) | 2000-03-15 |
CN1143132C true CN1143132C (zh) | 2004-03-24 |
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1998
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