CN101133317A - 经掺杂的二氧化硅微球光学离子传感元件 - Google Patents
经掺杂的二氧化硅微球光学离子传感元件 Download PDFInfo
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- CN101133317A CN101133317A CNA2006800035733A CN200680003573A CN101133317A CN 101133317 A CN101133317 A CN 101133317A CN A2006800035733 A CNA2006800035733 A CN A2006800035733A CN 200680003573 A CN200680003573 A CN 200680003573A CN 101133317 A CN101133317 A CN 101133317A
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Abstract
一种用于测定液体样品中目标离子浓度的传感元件,含有一种掺杂了下述物质的粒状二氧化硅:一种能够结合目标离子的离子载体;以及一种能够针对离子载体和目标离子的结合能够产生可检测信号的指示剂。可检测信号与液体样品中目标离子的浓度相关。
Description
交叉引用的相关申请
本申请要求2005年1月31日提交的美国临时专利申请No.60/648,527的优先权,其中全部内容引入本文以作参考。
关于联邦政府赞助研究或开发的声明
本发明是与美国政府合作,在美国国立卫生研究院颁发的DE14950的支持下完成的。在本发明中美国政府具有一定的权利。
技术领域
本发明涉及微球基化学传感元件,更具体说,涉及基于经掺杂的二氧化硅模板的微球光学离子传感元件。
背景技术
作为致力于传感元件微型化的一部分,微球基化学传感元件和生物传感元件的制造在过去的十年中正在受到越来越大的关注。微球传感平台具有很多优点。微米级别的传感元件可以在设定的局部环境下例如单细胞中询问被分析物的浓度。此外,所需的样品的容量小得多,从而可以提高灵敏度,缩短响应时间,相应降低试剂的成本,并且改进了检测的下限。此外,由于传感数据的高度冗余性,读出大量相同的微球可以提高精确度。微球基传感原理已经成功地被应用于各种读出形式,例如光纤显影和流式细胞计量术,例如,如题为“离子检测微球及其使用方法”的美国专利申请No.2004/0058384所述,其中全部内容引入本文以作参考。
在各种可以结合微球进行的分析测定中,中性载体基微球光极具有可靠的测试普通电解液离子活性的能力。可以通过各种方法制备离子或分子传感微球,包括如W.Seitz等人在Anal.Chim.Acta 400(1999)55以及Z.Shakhsher等人在Microchim.Acta 144(2004)147上所述的聚合物溶胀法。微球还可以通过如S.Peter等人在Anal.Chim.Acta 442(2001)25上所述的非均相聚合法、S.Shibata等人在Jpn.J.Appl.Phys.37(1998)41上所述的包含微粒模板的表面吸附法、以及I.Tsagkatakis等人在Anal.Chem.73(2001)315上所述的溶剂浇铸法进行制备。
如I.Tsagkatakis等人在Anal.Chem.73(2001)6083上所述的类似于美国专利4,162,282所公开的声响式铸造装置已经被用于在适度的、无反应活性的条件下大量制造可控尺寸的光学传感微球。制得的光学离子传感微球遵循经典的大容积光极理论,并且被用于测定Na+、K+、Ca2+、Pb2+和Cl-。最近,如S.Peper、A.Ceresa、Y.Qin、E.Bakker在Anal.Chem.Acta 500(2003)127上所述,在致力于避免增塑剂渗出的问题中,使用微粒铸造装置,开发出了基于甲基丙烯酸甲酯-甲基丙烯酸癸酯(MMA-DMA)共聚母体的用于K+的不含增塑剂的微球。
然而,上述方法存在如下的一个或多个缺点。制备微球所需要的设备要求特定的机械部件和附件,而这在大多数研究实验室中是无法获得的。产生的微球悬浮在水中,可能无法适应各种应用。微球的使用时间限定为小于6个月,更具体说为2~6周。这些早先的微粒的机械稳定性较差;在声波处理时可能会发生分裂,产生微末(碎片)从而干扰测定。此外,这些早先的微粒会彼此或与容器壁结合。当微粒在较高的局部浓度下储藏时更容易发生结合,这通常发生在微粒在储藏中产生沉积时。
二氧化硅微粒已广泛用于色谱柱中充填的固定相。如W.Pirkle等人在J.Org.Chem.44(1979)1957、以及N.Oi等人在J.Chromatogr.292(1984)427上所述,二氧化硅表面可进行化学改性以满足手性分离的需要。此外,如H.Figge等人在J.Chromatogr.351(1986)393上所述,二氧化硅表面可以涂布合适的聚合物以制成具有最佳分离特性的固定相。
如K.J.Albert等人在Anal.Chem.72(2000)1947上所述,含有染料的掺杂二氧化硅微粒被用于水蒸气检测,或如Y.Qin等人在Anal.Chem.75(2003)3038上所述被用于生物分子标记。然而,大多数掺杂二氧化硅微粒的使用仅限于单组分传感系统,因此不适用于离子传感的用途。
因此,需要一种改进的离子传感微球。
发明内容
因此,本发明的目的在于一种在液体样品中检测目标离子浓度的传感元件,该传感元件包含:掺杂有能够与样品中的目标离子结合的离子载体、和能够针对离子载体与目标离子的结合而产生可被检测的信号的指示剂的粒状二氧化硅。可检测的信号与液体样品中的离子浓度有关。指示剂可以是有色离子载体。
传感元件还可以具有内增塑聚合物。传感元件可任选地含有支承聚合物及增塑剂。支承聚合物可以是PVC而增塑剂可以是二(2-乙基己基)癸二酸酯(DOS)。
粒状二氧化硅可以是球形或其他三维的形状。粒状二氧化硅可选被硅烷化。传感元件还可以包含亲脂性的阳离子交换剂。亲脂性的阳离子交换剂可以是四(3,5-二(三氟甲基)-苯基)硼酸钠(NaTFPB)。
本发明的另一个目的是使用传感元件测定液体样品中的离子的方法。由含水的硅胶微球制成的传感元件可选被干燥,从而制得干燥的传感元件用于将其储藏到使用前。可以将干燥的传感元件再悬浮化以制得再悬浮化的传感元件并且用于检测。传感元件可任选地通过流式细胞计数器以测定可以检测的信号。
该传感元件也可以用于光纤束。
附图说明
根据下列描述、附加的权利要求书和附图,可以更好地理解本发明的特性、各方面以及优点,其中:
图1a所示为用于本发明的在用传感成分掺杂之前的硅烷化的二氧化硅颗粒的扫描电子显微镜图;
图1b所示为图1a中的颗粒掺杂了实施例Na-J的传感成分后的扫描电子显微镜图;
图2所示为由实施例Na-J的单二氧化硅基Na+选择性微球光学传感元件与(A)10-2M HCl和(B)10-2M NaOH接触时观察到的空间分辨荧光光谱的三维图;
图3a所示为由荧光显微术所表征的根据实施例Na-J的微球在pH值7.4时的响应图;
图3b所示为由荧光显微术所表征的根据实施例Ca-L的微球在pH值7.4时的响应图;
图4所示为含有各种被标准化至pH值为7.4的离子载体的光极由实验所得的选择性系数的表格;
图5a所示为由分解的流式细胞计量术所表征的根据实施例Na-J的微球在pH值7.4时的响应图;
图5b所示为由分解的流式细胞计量术所表征的根据实施例Ca-L的微球在pH值7.4时的响应图;
图6所示为根据实施例Na-H的Na+选择性微球在光纤束被蚀刻的凹处上观察到的空间分布的显微照相图;以及
图7所示为根据实施例Na-H的五个临近的Na+选择性微球在光纤束被蚀刻的凹处上观察到的荧光光谱的三维图。
具体实施方式
根据本发明的一个实施方式,目的在于基于掺杂粒状二氧化硅模板的离子选择性光学传感元件,及其制备和使用方法。本发明也是Analytica Chimica Acta,537,29April 2005,pp.135-143上题为“基于掺杂硅胶模板的微球光学离子传感元件”的文献的主题,其中全部内容引入本文以作参考。
该传感元件优选由具有多孔的二氧化硅基质的微球构成。在本发明的一个实施方式中,二氧化硅基质为购自瑞典EKA化学公司的平均粒径大约为3.5μm的球形二氧化硅Kromasil100。其中,其他可以用于本发明的二氧化硅基质包括具有合理紧凑的尺寸分布的其他球形二氧化硅,例如直径为5、7、10、13或16μm的Kromasil 100。
该传感元件具有能够与液体样品中的目标离子结合并且对其具有较高选择性的离子载体。该传感元件可以连同各种用于检测不同目标离子的离子载体使用。该离子载体的例子包括但不限于对于目标离子例如氢、Li+、Na+、K+、Ca2+、或Mg2+、或金属离子例如Pb2+、Cu2+、Hg2+、Ag+、以及氧化物例如UO2 2+具有选择性的离子载体。
在本发明的一个实施方式中,离子载体为叔丁基杯[4]芳烃四乙酯(钠离子载体X)。在本发明的另一个实施方式中,离子载体为Ca2+离子载体AU-1嫁接在聚(丙烯酸正丁酯)中。离子载体的浓度可以是由大约0.1到大约200mmole/kg,并且优选为由大约10到大约50mmole/kg。
本发明可以使用的其他离子载体包括例如:钾离子载体I(缬安霉素)、钾离子载体II(二((苯并-15-冠-4)-4’-基甲基)庚二酸酯)、钾离子载体III(BME44;(2-十二烷基-2-甲基-1,3-丙二基-二(N-(5’-硝基(苯并-15-冠-5)-4’-基)氨基甲酸酯))、氯离子载体I(5,10,15,20-四苯基-21氢,23氢-卟吩锰(III)氯化物;Mn(III)TPPCl)、氯离子载体II(ETH 9009;(4,5-二甲基-3,6-二辛氧基-1,2-二苯撑)-二-(三氟乙酸汞))、钠离子载体I(ETH 227;N,N’,N”-三庚基-N,N’,N”-三甲基-4,4’,4”-次丙基三(3-氧杂丁酰胺))、钠离子载体II(ETH 157;N,N’-二苄基-N,N’-二苯基-1,2-亚苯基二氧二乙酰胺)、钠离子载体III(ETH 2120;N,N,N’,N’-四环己基-1,2-亚苯基二氧二乙酰胺)、钠离子载体IV(DD-16-C-5,2,3:11,12-二萘烷(Didecalino)-16-冠-5)、钠离子载体V(ETH 4120;4-十八酰氧甲基-N,N,N’,N’-四环己基-1,2-亚苯基二氧二乙酰胺)、钠离子载体VI(二((12-冠-4)甲基)十二烷基甲基丙二酸)、钠离子载体VIII(二((12-冠-4)甲基)2,2-二(十二烷基丙二酸))、钠离子载体X(4-叔丁基杯[4]芳烃-四乙酸四乙酯)、钙离子载体I(ETH1001;(-)-(R,R)-N,N’-(二(11-乙氧基羰基)十一烷基)-N,N’-4,5-四甲基-3,6-二氧辛二酰胺;二乙基-N,N’-((4R,5R)-4,5-二甲基-1,8-二氧代-3,6-二氧杂亚辛基)-二(12-甲基氨基-十二酸酯))、钙离子载体II(ETH 129;N,N,N’,N’-四环-3-氧杂戊二酰胺)、钙离子载体III(A23187;钙霉素)、钙离子载体IV(ETH 5234;N,N-二环己基-N’,N’-二(十八烷基)-3-氧杂戊二酰胺),以上离子载体均购自Fluka(密尔沃基,威斯康星州)。
该传感元件还包含能够在应答离子载体与目标离子的结合中产生可被检测信号的指示剂。在本发明的一个实施方式中,指示剂为有色离子载体。有色离子载体可以定量和/或检测样品中的目标离子。当通过与离子载体结合的目标离子交换质子时有色离子载体发生去质子化,并且有色离子载体的质子化的改变会造成其光学特性的可测性的改变。
有色离子载体可以是例如9-(二乙基氨基)-5-十八酰亚氨基-5氢-苯并[a]吩恶嗪(有色离子载体I,ETH 5294)。本发明可以使用的其他指示剂包括例如有色离子载体II;ETH 2439;9-二甲基氨基-5-(4-(16-丁基-2,14-二氧代-3,15-二氧杂二十烷基)苯基亚氨基)苯并[a]吩恶嗪、有色离子载体VI;ETH 7075;4’,5’-二溴荧光素十八烷基酯、以及有色离子载体III;ETH5350;9-(二乙基氨基)-5-((2-辛基癸基)亚氨基)苯并[a]吩恶嗪。
该传感元件还可以包含内增塑聚合物例如聚(丙烯酸正丁酯)或甲基丙烯酸甲酯(MMA)和甲基丙烯酸癸酯单体的共聚物(如2002年12月5日提交的美国专利申请号No.10/313,090所述),其中全部内容引入本文以作参考。
此外,该传感元件还包括支承聚合物和增塑剂。支承聚合物可以是例如高分子量聚氯乙烯(PVC)。增塑剂可以是例如购自Fluka(密尔沃基,威斯康星州)的二(2-乙基己基)癸二酸酯(DOS)。其他的增塑剂包括二(2-乙基己基)邻苯二甲酸酯和2-硝基苯基辛基醚。
本发明的传感元件还可以包括其他添加剂,例如离子交换剂,以促进目标离子从样品上脱出以及目标离子向离子载体的迁移。离子交换剂优选为亲脂性的阳离子交换剂。亲脂性的阳离子交换剂可以是例如购自美国Dojindo分子科技公司的四(3,5-二(三氟甲基)-苯基)硼酸钠(NaTFPB)。
其他阳离子交换剂包括卡巴(carba)-闭环十二硼酸酯、尤其是卤化碳硼烷阴离子。卤化碳硼烷阳离子交换剂的例子包括三甲基铵-2,3,4,5,6,7,8,9,10,11,12十一溴碳硼烷(TMAUBC)(参见美国专利申请号No.10/313,090)、以及十一氯化碳硼烷(UCC)、六溴化碳硼烷(HBC)和十一碘化碳硼烷(UIC)阴离子的盐(例如三甲基铵盐)。
下面对根据本发明的一个实施方式的微球的制备进行描述。如K.Wygladacz等人在Sens.Actuators B 83(2002)109、以及M.E.McGovern等人在Langmuir(1994)360上所述,掺杂前可以进行硅烷化以提高二氧化硅颗粒的疏水性,并且其中全部内容引入本文以作参考。优选将二氧化硅模板小心地密封在瓶中或其他容器中,并且保持在真空中以除去孔内的空气。然后在二氧化硅颗粒中掺杂适当的传感成分。
将包括离子载体和指示剂的传感成分溶解于适当的溶剂例如四氢呋喃(THF)中,并且与二氧化硅模板缓和地混合。然后用例如铝箔覆盖混合物,随后优选将其置于黑暗中一直到在溶剂蒸发时传感成分进入多孔的二氧化硅模板中。在使用前优选将制得的微球放置在黑暗中保持干燥。
微球光学传感元件优选用阳离子交换剂(“R-”),离子载体(“L”)和H+-选择性有色离子载体(“Ind”)掺杂。对于这种大容积光极,将带(“z+”)电荷的阳离子分析物(“Iz+”)从水溶液中萃取至有机传感相中,从而通过下列阳离子交换机理消除氢离子:
I2+(水相)+nL(有机相)+zIndH+(有机相)+zR-(有机相)
=Ln z+(有机相)+zInd(有机相)+zR-(有机相)+zH+(水相)(1)
结合电荷和质量平衡,并且定义质子化“Ind”的摩尔分数为(“1-α”),分析物的活性可以表达为:
在pH值已知的缓冲溶液中,可以通过有色离子载体的质子化的程度(“1-α”)测定分析物离子的活性,(“1-α”)可基于所观察到的对于有色离子载体的质子化的(“Rpro”)和未质子化形式的(“Rdep”)发射强度进行计算:
在下文中将参考实施例进行讨论,荧光显微法和流式细胞计量术可以显示出本发明的微球光学传感元件的响应遵循经典的大容积光极理论。检测范围与生理电解液的级位一致,并且由此所获得的选择性数据与由那些不含二氧化硅的传感颗粒所获得的选择性数据类似。此外,本发明的微球光学传感元件如果以干燥形态储藏则具有大于6个月的保存期限。
实施例
Ca
2+
离子载体AU-1的制备
根据Y.Qin、S.Peper、A.Radu、A.Ceresa、E.Bakker在Anal.Chem.75(2003)3038上所述的方法合成Ca2+离子载体N,N-二环己基-N’-苯基-N’-3-(2-丙酰)氧苯基-3-氧杂戊二酰胺(AU-1),其中全部内容引入本文以作参考,并且如下所述将2%(w/w)和5%(w/w)的AU-1接枝于聚(丙烯酸正丁酯)。丙烯酸正丁酯购自Polysciences(沃灵顿,PA)。
首先,使用下述方法合成AU-1。步骤1:向二甘醇酐(1.16g,10mmol)在100mL的无水二氯甲烷中的经搅拌的溶液中加入二环己胺(3.62g,20mmol)。将混合物在室温下搅拌3小时。然后,向反应混合物中加入20mL 6N的HCl。将固体过滤去,分离滤液中的有机层并且用无水硫酸钠进行干燥。使用旋转蒸发器除去二氯甲烷。从乙酸乙酯中重结晶出3-氧杂戊酸-N,N’-二环己酰胺(I)的白色晶体,其产率为92%(2.74g)。步骤2:在N2保护下将3-羟基二苯胺(3.33g,18mmol)溶解于25mL的无水THF中。接着,将三乙胺(1.98g,19.5mmol)加入上述溶液。然后,在N2气氛中在-5℃下使用注射器将丙烯酰氯(1.62g,18mmol)逐滴加入反应混合物。25分钟后,加入30mL的饱和NaHCO3溶液以中止反应。然后分离有机相,并进行水洗。将溶剂蒸发后,使用快速色谱法(1∶1EtOAc/己烷)纯化粗制产物。从而制得了浅黄色固体间-苯胺基苯基丙烯酸酯(II),其产率为60%(2.88g)。步骤3:在室温下边搅拌边向I(0.736g)和II(0.529g)在30mL的无水CH2Cl2中所形成的溶液中加入Et3N(0.8g)。然后加入0.612g的BOP-Cl。将混合物回流24小时。使用10mL的饱和NaHCO3和水洗涤反应混合物。分离并蒸发溶剂后得到有机相。使用快速色谱法(1∶5EtOAc/己烷)纯化剩余物。从而得到了浅黄色固体(C31H38N2O5,MW 518.65),其产率为50%。
通过热引发自由基溶液聚合合成结合了AU-1的聚合物。对含有丙烯酸正丁酯(1g)和适量的离子载体AU-1(2或5wt.%)的乙酸乙酯溶液用N2吹扫10分钟,然后加入5.1mg的聚合引发剂偶氮二异丁腈(98%(AIBN),购自Aldrich(密尔沃基,威斯康星州))。持续搅拌该均相溶液,并将温度设为90℃,并保持16小时。
反应结束后,将溶液蒸发,并且将聚合物重新溶于10mL的二恶烷。在剧烈搅拌下将聚合物溶液的等分试样(2mL)加入100mL的蒸馏水中。收集白色沉淀并且溶于25mL的二氯甲烷,然后用无水Na2SO4脱水并过滤。将溶液蒸发,并将获得的透明聚合物在实验室环境条件下进行干燥。从而合成了两批接枝于聚(丙烯酸正丁酯)的不同浓度的离子载体AU-1:(1)2wt.%的AU-1(38.6mmol/kg)和(2)5wt.%的AU-1(96.5mmol/kg)。
微球的制备
使用下列方法制备多种不同类型的微球。在掺杂前先进行硅烷化。使用甲苯清洗平均粒径大约为3.5μm的球形二氧化硅颗粒Kromasil 100以除去杂质,抽真空以除去空气,并且在平底反应器中与3-(三甲氧基甲硅烷基)丙基甲基丙烯酸酯(10%,v/v,在甲苯中)混合。使用水回流将温度在60~70℃下保持3~4小时。随后除去过量的反应物和溶剂,并且清洗硅烷化的微球并继续抽真空。
然后用合适的传感成分掺杂硅烷化的二氧化硅模板,总质量为20mg(掺杂成分+二氧化硅模板)。在称重前后将二氧化硅模板小心地密封在瓶中并且保持真空,以除去微孔中的空气。将传感成分溶于THF并且缓和地与二氧化硅模板混合。然后用155铝箔覆盖混合物,并且在黑暗中保持72小时。同时在溶剂蒸发时使传感成分进入多孔的二氧化硅模板。在表征前将制得的微球放置在黑暗中保持干燥。
钠选择性微球
将由40mmol/kg的钠离子载体(X)、10mmol/kg的ETH 5294、20mmol/kg的NaTFPB和多种组成的DOS(10、20、30、40、50%,w/w)组成的Na-A~Na-E型分别与适量的二氧化硅模板(17.1、15.1、13.1、11.1或9.1mg)混合。经改性的组合物(同样是20mg的总质量)由如上所述同样浓度的钠离子载体(X)、ETH 5294、NaTFPB组成,除了DOS被分别替换为5或10%的聚(丙烯酸正丁酯)(Na-F和Na-G型)或5wt.%的PVC(Na-H型)或(5wt.%的PVC+10wt.%的DOS)(Na-I型)。Na-J型含有39.3mmol/kg的钠离子载体(X)、9.7mmol/kg的ETH 5294、19.1mmol/kg的NaTFPB、2wt.%的PVC和10wt.%的DOS,以及17.6mg的二氧化硅模板(总质量20mg)。
钙选择性微球
用离子载体Ca(IV)掺杂的微球
使用N,N-二环己基-N’,N’-二(十八烷基)-3-氧杂戊酰胺(Ca离子载体IV,ETH 5234)(10.9、21.5、39.0或48.9mmol/kg)、ETH 5294(2.0、4.1、5.0或6.2mmol/kg)、NaTFPB(3.4、6.0、7.5或9.1mmol/kg)和10%(w/w)的DOS分别掺杂16.0、15.5、15.1和14.7mg的二氧化硅模板制得Ca-A、B、C和D型。
Ca-E~Ca-G型含有39.0mmol/kg的Ca(IV)离子载体、5.0mmol/kg的ETH 5294、7.5mmol/kg的NaTFPB、以及5或10wt.%的聚(丙烯酸正丁酯)(Ca-E和Ca-F型)或5wt.%的PVC(Ca-G型)。
用接枝于聚(丙烯酸正丁酯)的AU-1离子载体掺杂的微球
Ca-H~Ca-K型具有2wt.%的接枝于聚(丙烯酸正丁酯)的AU-1。Ca-H~Ca-K型分别具有占总质量的15、30、40或50%(w/w)的聚合物,所述聚合物转化成5.8、11.6、15.4和19.3mmol/kg的AU-1;0.6、1.2、3.0或3.6mmol/kg的ETH 5294;1.1、2.3、4.6或5.8mmol/kg的NaTFPB;以及10wt.%的DOS和17.4、14.3、9.7或8.0mg的二氧化硅模板。
Ca-L型具有5wt.%的接枝于聚(丙烯酸正丁酯)的AU-1。最近AU-1被接枝于MMA-DMA共聚物基质以制备无增塑剂的离子传感系统例如离子选择性膜和光极薄膜。参见Y.Qin、S.Peper、A.Radu、A.Ceresa、E.Bakker在Anal.Chem.75(2003)3038所述。如先前Heng和Hall所报道,聚(丙烯酸正丁酯)是一种有用的内增塑聚合物,L.Heng、E.Hall发表于Anal.Chem.72(2000)42。Ca-L型具有16wt.%的聚合物(30.1mmol/kg的Ca2+离子载体AU-1)、4.2mmol/kg的ETH 5294、8.0mmol/kg的NaTFPB和10wt.%的DOS,掺杂于11.9mg的硅烷化的二氧化硅模板。
实施例测试
在PARISS图像光谱仪(Light Form,Belle Mead,NJ)上结合Nikon Eclipse E400显微镜[15]进行荧光显微测试。系统配备了两个EDC 1000L CCD相机(Electrim公司,普林斯顿,新泽西州)以及一个外延荧光汞灯(Southern Micro Instruments,GA),另外还有一个由Pariss光谱图像软件(Light Form)控制的电动载物台(Prior Optiscan ES9,Fulbourn,Cambs,U.K.)。对于所制得微球的表征和分辩光谱,将一块Nikon Plan Fluro 40×0.75的物镜和一个EX510~560nm的滤光镜结合使用。曝光时间选择200~600ms以满足荧光强度。
微球被置于缓冲样品溶液中,并放置在黑暗中保持20~40分钟。10毫摩尔的HCl或10mM的NaOH被分别用于在完全质子化或去质子化的状态下记录光谱。随机选择6~10个微球以记录光谱。通过计算ETH 5294在645和675nm处的两个荧光强度峰的比例可以获得质子化的程度。
使用通过将标准激光替换为635nm的二极管激光并且配置了对测定波长范围在650~675nm内的荧光具有选择性的滤光镜和检测器的经改进的Beckman Coulter EPICS XL流式细胞仪进行流式细胞测试。使用650nm的长通发射滤光镜和660(±15)nm的通带滤光镜收集在650~675nm之间发射的荧光。将该硅胶基微球浸入缓冲样品溶液20~30分钟,以使其平衡。
在5kV下使用Zeiss DSM 940扫描电子显微镜,根据I.Tsagkatakis等人在Anal.Chem.73(2001)6083上具体描述的方法,获得二氧化硅模板和掺杂微球传感元件的SEM图像,其中全部内容引入本文以作参考。在测试前,将干燥的微球沉积在铝棒上并且喷涂10~20nm的Au/Pd大约60秒。
样品缓冲溶液
使用纳米纯化去离子水配制所有的缓冲溶液(18.2MΩcm)。分别在10-3M的Tris(三(羟甲基)氨基甲烷,购自Fluka(密尔沃基,威斯康星州))或10-2M的MES中配制10-5~1M的NaCl或CaCl2,并且对于Tris将pH值调至7.4(或对于MES调至5.5)。如M.Lerchi等人在Anal.Chem.64(1992)1534上以及E.Bakker等人在Anal.Chem.64(1992)1805上所详述的,对于选择性测试使用分液法,其中全部内容引入本文以作参考。
大容积光极的选择性测试需要有对所包含的每种离子的完整的校准曲线。这些曲线是对于各种试样在相同的pH值下用相同的薄膜组合物获得的理想曲线。各个校准提供了所有被测物离子的有关热力学交换和共萃取常数和化学计算等性状的最精确的信息。
可以获得对于任何干扰离子的初级选择性系数的各个离子的响应曲线可以通过E.Bakker等人在Anal.Chem.64(1992)1805上所述的公式27和36进行作图并计算。
在pH值为7.4并且含有1M的一种干扰离子盐的10-3M的Tris缓冲液中记录响应。对于基于二氧化硅模板的Na+选择性微球,被测定的干扰阳离子为K+、Mg2+和Ca2+,而对于Ca2+选择性微球,测定的为K+、Na+和Ma2+。
测试结果
Na
+
选择性微球
使用增塑剂DOS制得的Na-A型微球定性地响应于Na+活性的变化。然而颗粒间的偏差和与理论预期的响应行为之间的偏差很大,并且24小时后,在显微镜下可以检测到被增塑组分的浸出。在其他组合物Na-B~Na-E中,都使用了不同浓度的增塑剂DOS,随着增塑剂含量的增大被增塑组分的实际浸出的程度也会增大。
在Na-F~Na-J型中,其中的增塑剂DOS被替换为聚(丙烯酸正丁酯)或向混合配方中加入了PVC,结果显示如Na-J型加入PVC会大幅提高制得的微球的响应特性。图2所示为具有染料的Na-J型二氧化硅基Na+传感微球在其被完全质子化(10-2M的HCl)和去质子化(10-2M的NaOH)形式下的3D响应光谱,所述的二种形式其峰的形状都类似于PVC基微球的。
这证实了有色离子载体掺杂进入二氧化硅模板后的基本功能。染料ETH 5294(有色离子载体I)是H+选择性有色离子载体,它在掺杂二氧化硅模板中具有在645nm(去质子化)和675nm(质子化)的双荧光最大发射。通过获得这两个峰的强度比,使用公式(2)可以计算出有色离子载体的质子化程度。比值测定的优点在于可以获得可靠的信号据而减少了图像漂白的危害,并且减少了光源不稳定和微球尺寸变化的影响。
图3A所示为用荧光显微法表征的Na-J型微球在pH值7.4时相应的Na+响应曲线与相关的的选择性的图。图上的数据点为平均实验值,并且错误栏指的是由5~10个独立测试所观察到的标准偏差。
使用实验组合物由公式(2)推导出理论曲线。对于理论曲线的合适的离子交换常数(公式(2)中的Kex)和对于不同传感系统对普通干扰离子的选择性系数如表4所示,并且与通过声响式微粒铸造仪制得的不含二氧化硅的PVC-DOS颗粒所获得的数据相比较。
本发明的微球对于K+、Ca2+和Mg2+具有与通过声响式微粒铸造仪制得的不含二氧化硅的PVC-DOS颗粒大致相同的选择性。对于由二氧化硅模板制得的微球,测试范围适用于直接测试人类唾液(受激,pH值为7.0~7.5,Na+典型为4.3~28mM)。由二氧化硅模板制得的微球也可以被用于测定10倍稀释的人类血浆(pH值为7.4,Na+为135~150mM)。
Ca
2+
选择性微球
在组合物不同的含有离子载体Ca(IV)、ETH 5294、NaTFPB和DOS的Ca-A~Ca-D型微球中没有观察到适当的钙响应。在Ca-E~Ca-G型中,使用了不同的基质,包括聚(丙烯酸正丁酯),或者加入了PVC,观察到的钙的响应仍然不能令人满意。
在Ca-H~Ca-K型微球中,使用接枝于聚(丙烯酸正丁酯)上的AU-1(2%,w/w),可以发现结果有色离子载体的官能浓度过低,无法可靠地进行荧光显微法测定。进一步提高接枝于离子载体的浓度会造成微球严重积聚。
在Ca-L型微球中,在聚(丙烯酸正丁酯)聚合物中AU-1的浓度为5%(w/w),可以观察到对Ca2+的选择性很好。图3B所示为对于Ca-L型在pH值为7.4时观察到的Ca2+的响应以及根据公式(2)的理论校准曲线。
在生理样品中观察到的Ca-L型微球对普通干扰阳离子的选择性与从薄光极薄膜获得的数据一致(参见图4)。由于Na+的含量非常大所以是重要的干扰,对于Na+的选择性超过三个数量级。
当有色离子载体半质子化时(α=0.5),在pH值为7.4时Ca2+活性相应为~1mM,说明测定范围适合直接测定pH值为7.4的人类血浆中的Ca2+(1~1.2mM),或pH值为7.0~7.5的受激人类唾液(0.8~2.8mM)。
对于制得的基于掺杂硅胶模板的微球,可以观察到典型的平衡时间大约为10min,与常规的经塑化的PVC微粒相比平衡时间稍长,但短于MMA-DMA基微粒。掺杂了经接枝的AU-1的钙选择性光学传感微球与使用易溶的离子载体的钠选择性微球相比显示了更长的平衡时间(大约25分钟)。
微球的流式细胞计量术分析
流式细胞计量术适用于基于增塑PVC的荧光微球光学传感元件的表征,其中记录了有色离子载体ETH 5294的去质子化形式的单参数柱状图以检测荧光的变化。流式细胞计数器和荧光显微镜都被应用于制得的微球的表征。虽然流式细胞计数器不能如同荧光显微镜那样空间分辩或光谱分辩单个微粒的荧光,但可以提供大量微粒的统计行为的信息。
在FL1通道(具有650nm的长通滤光镜和660nm的通带滤光镜(±15nm))的柱状图中,观察到的计数的数量对峰通道荧光的对数值作图,并且由累计微粒计数的数量产生高斯(Gaussian)形的曲线。随着样品离子的浓度增大,会发现ETH 5294的去质子形式的荧光强度也随之增大,并且造成高斯曲线的峰移位,反映出离子响应。大约10,000个微球的整个柱状图的系数变化(CV)范围在7.13~29.33,这比通过声响式铸造法制得的常规PVC微粒中所观察到的更大,表明尺寸的再现性较差,而且这受原始硅胶模板的尺寸分布限制。
图5A和5B所示为通过流式细胞计数器获得的钠(Na-H型)或钙选择性(Ca-L型)微球的校准曲线和相关的选择性数据。假设了制得的微球的光极性状,使用公式(4),质子化的程度(“1-α”)为单参数柱状图中FL1通道的荧光峰位置(P)所示:
对于图5A中的钠响应的理论曲线(公式(2)),logKex为-5.6,并且对于钙,logKex为-9.1。这些数据与通过荧光显微镜在单个微粒上所获得的数据相似(Na+的logKex=-5.0,Ca2+的logKex为-9.0,参见图4)。图4中还显示了电位干扰的实验选择性系数(log kOsel I,J的值)。测试范围也与荧光显微镜法接近。总之,通过流式细胞计量术和荧光显微镜法获得的响应和选择性数据可以互相很好地对应,并且两种技术都适用于表征本发明的微球。
二氧化硅基微球对于光纤光学传感元件阵列的应用
常规的PVC光学离子传感微球已经被成功地应用于能够对数万传感元件的响应进行平行检测的光纤束。光纤束已经被发现并作为这一功能的平台,主要因为它们可以简易被蚀刻从而形成高度一致性的与所制得微球传感元件构成尺寸吻合的“凹处”。通过将光纤束的传感端浸泡在不同浓度的分析物缓冲液中,可以从光纤束的另一端获取有色离子载体荧光光谱并使用荧光显微镜加以表征。在一条直径为2mm的光纤束上,有大约3500条其纤心直径为约4.6μm的独立光纤丝。
对不同离子具有选择性的经增塑的PVC微球已任意沉积在同一条光纤束上,从而实现多重光学传感。作为可以替代经增塑的PVC微球,根据本发明的基于掺杂硅胶微粒的离子检测微球能够沉积在光纤束的被蚀刻的末端。
根据本技术领域内已知的方法将六棱的光纤束进行打磨、清结、侵刻蚀并且经声波处理,如J.R Epstein等人在Biosens.Bioelectron 18(2003)541上所述,其中全部内容引入本文以作参考。将根据样品Na-H制得的Na+选择性微球与去离子水混合,并将1μm的部分悬浮混合物放置于光纤束的被蚀刻的凹处端。当微球沉淀入凹处中后,使用去离子水洗去过剩的微粒。然后,在获得光谱响应前,将光纤束的被蚀刻端浸入10-2M的HCl中20分钟。
如图6所示,在荧光模式下观测到90%的微粒覆盖在光纤束上。所制得微球的直径(约为3.5μm)与经蚀刻的凹处的尺寸(约为4.6μm)相适合。图7所示是从光纤束的经蚀刻的凹处中所发现的五个Na+选择性微球(样品Na-H)的被观察到的三维荧光光谱图。相同的形状和接近的光亮度值显示了相邻的微球的荧光光谱具有很好的复现性。
根据本发明的微球满足许多规范并成功地应用于生理样品,包括可靠的离子响应和对于普通干扰离子的选择性。二氧化硅模板的存在并没有影响传感化学,并且微球的响应体现了大容积光极的传感原理。检测到的响应与从光极薄膜以及声响式铸造的聚合物微球获得的结果相当。
此外,本发明的微球不需要如在常规PVC基微球情况下的熟化过程。本发明的微球在进行掺杂后可以立即使用。由于其具有高密度,本发明的微球可以被离心分离,并且无论干燥或在溶液中都易于处理。
当本发明的微球在被密封并在黑暗环境中保持干燥时可以保存六个月以上。然后可以将微球重新悬浮以制得重新悬浮的复合物。在对Na-H和Ca-L型微球掺杂六个月后再次进行流式细胞计量术进行测量,发现最后所得到的响应复现了最初的测量。
通过以上对本发明的描述,显然可以在不背离之前的叙述以及之后的权利要求书中所述的容本发明的范围和明晰的含义下可进行许多修改和改进。
尽管已经采用优选的形式对本发明进行了详尽描述,但也可以使用其他方式。因此,附加的权利要求书的精神和范围并不受以上优选所述内容的限制。除了至少一些互斥的特征和/或步骤的组合之外,说明书中公开的所有特征,包括权利要求书、说明书摘要、附图和公开的任何方法或过程中的所有步骤,均可以任意组合形式进行组合。除非另有明确的指出,说明书中公开的每个特征,包括权利要求书、说明书摘要和附图,可以用起相同、相当或相似作用的特征所取代。因此,除非另有明确的指出,公开的每个特征仅仅是非特殊系列中相当或相似的特征的一个例子。
权利要求书中的任何部分,凡是没有标明“方法”用于实现特定功能或“步骤”用于实现特定功能的,均不应理解为在35U.S.C§112中规定的“方法”和“步骤”条款。
Claims (19)
1.一种用于测定液体样品中目标离子浓度的传感元件,含有粒状二氧化硅,所述二氧化硅掺杂有:
一种能结合目标离子的离子载体;以及
一种针对离子载体和目标离子间的结合能够产生可检测信号的指示剂;
其中所述可检测信号与液体样品中目标离子浓度相关。
2.如权利要求1所述的传感元件,其特征在于,所述指示剂为有色离子载体。
3.如权利要求1所述的传感元件,其特征在于,所述离子载体为叔丁基杯[4]芳烃四乙酯。
4.如权利要求1所述的传感元件,其特征在于,所述传感元件进一步含有内增塑聚合物。
5.如权利要求4所述的传感元件,其特征在于,所述内增塑聚合物为聚(丙烯酸正丁酯)。
6.如权利要求4所述的传感元件,其特征在于,所述离子载体为接枝于内增塑聚合物的AU-1。
7.如权利要求1所述的传感元件,其特征在于,所述传感元件进一步包含支承聚合物和增塑剂。
8.如权利要求7所述的传感元件,其特征在于,所述支承聚合物为PVC,所述增塑剂为DOS。
9.如权利要求1所述的传感元件,其特征在于,所述粒状二氧化硅为球形颗粒。
10.如权利要求1所述的传感元件,其特征在于,所述粒状二氧化硅被硅烷化。
11.如权利要求1所述的传感元件,其特征在于,所述传感元件进一步包含亲脂性阳离子交换剂。
12.如权利要求11所述的传感元件,其特征在于,所述亲脂性阳离子交换剂为NaTFPB。
13.一种检测液体样品中目标离子的方法,所述方法包含下列步骤:
a)使多个如权利要求1所述的传感元件与液体样品接触,从而使离子载体与样品中的目标离子结合;以及
b)测定可检测的信号。
14.如权利要求13所述的方法,其特征在于,所述测定可检测的信号的步骤包含使所述传感元件通过流式细胞计数器。
15.一种用于检测液体样品中离子浓度的传感元件的制备方法,包含下列步骤:
a)获得大量硅胶微球,所述硅胶微球含有水;
b)对所述微球掺杂离子载体和指示剂以制得传感元件的悬浮液,所述离子载体能够结合样品中的离子,所述指示剂针对离子载体和目标离子的结合能够产生可检测的信号;以及
c)干燥所述传感元件以基本上除去所有的水。
16.如权利要求15所述的方法,其特征在于,进一步包含再次使已干燥的传感元件悬浮以制得再次悬浮的传感元件的步骤。
17.一种用于检测液体样品中离子浓度的装置,包含:
a)一种具有多个纤维的光纤束,每个纤维具有带凹陷的样品端;以及
b)多个微粒,每个微粒包含:掺杂了能够结合样品中离子的二氧化硅基质;和针对离子载体和目标离子的结合能够产生可检测信号的指示剂;
其中所述可检测信号与液体样品中目标离子的浓度相关;以及
多个微粒位于纤维的凹陷处中。
18.一种用权利要求15所述的方法制得的传感元件,用于检测液体样品中离子的浓度。
19.一种用权利要求16所述的方法制得的传感元件,用于检测液体样品中离子的浓度。
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IL154960A0 (en) * | 2000-10-10 | 2003-10-31 | Du Pont | Polymers having attached luminescent metal complexes and devices made with sych polymers |
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-
2006
- 2006-01-31 WO PCT/US2006/003546 patent/WO2006083960A1/en active Application Filing
- 2006-01-31 EP EP06720074A patent/EP1864114A1/en not_active Withdrawn
- 2006-01-31 JP JP2007553375A patent/JP2008529014A/ja active Pending
- 2006-01-31 CN CNA2006800035733A patent/CN101133317A/zh active Pending
- 2006-01-31 US US11/345,553 patent/US20060240560A1/en not_active Abandoned
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108020589A (zh) * | 2016-10-28 | 2018-05-11 | 中国科学院烟台海岸带研究所 | 一种海水中钙离子的检测方法 |
CN108020589B (zh) * | 2016-10-28 | 2021-02-26 | 中国科学院烟台海岸带研究所 | 一种海水中钙离子的检测方法 |
Also Published As
Publication number | Publication date |
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US20060240560A1 (en) | 2006-10-26 |
JP2008529014A (ja) | 2008-07-31 |
EP1864114A1 (en) | 2007-12-12 |
WO2006083960A1 (en) | 2006-08-10 |
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