若干新型高灵敏度荧光检测系统的构建与评价
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摘要
高分离度与高检测灵敏度是液相色谱追求的永恒目标。得益于各种先进分离和检测手段的发展,多数样品已经能够得到良好的分离分析,但是对于超低含量物质的分析,检测灵敏度的提高仍具有极大的挑战性。荧光检测法具有高选择性和高灵敏度的特征,与高效液相色谱(HPLC)和毛细管电泳(CE)方法联用可充分结合两者的优势,是复杂样品分析的有利工具。
采用473nm固体激光器和365nm、335nm发光二极管(LED)为光源,构建了多种HPLC用荧光检测器。将激光诱导检测器(LIFD)与HPLC结合构建成新型HPLC-LIFD系统。基于精密三维调节架的使用及光路优化设计,LIFD对FITC的最低检测浓度达到10-12mol/L。使用该系统分析水中微量生物胺,亚精胺、腐胺和组胺的检测限分别为0.13、0.22和0.23(?)nmol/L,回收率：95%-105%。进一步将该系统用于不同类型茶叶茶水中荧光物质及胺类物质的分析,共检测到15-35荧光物质和48种以上胺类物质,也揭示了不同茶叶茶水中荧光物质和胺类物质种类和含量上的差异。
基于铕离子(Eu)荧光探针的窄范围发射光谱和大斯托克斯位移优势,设计通用的模块化共聚焦光路系统,构建了铕离子荧光探针专用HPLC-LED-IFD分析系统。对Eu荧光探针BHHCT-Eu衍生的BSA进行分析,检测限达到10-8mol/L。考察了Eu荧光探针衍生化物质在不同模式HPLC中的分离特征,证实BHHCT-Eu衍生化蛋白质可在反相色谱模式下操作。通过置换LED光源和滤光片,也搭建了适用于苯并检测的365nm LED-IFD。
充分考虑中空纤维透析膜的截留大分子及小分子置换的特征,设计了一种新型高效毛细管电泳蛋白质柱后衍生膜反应器,并将之与CE系统和LIFD结合,构建了基于蛋白质柱后衍生膜反应器的CE-LIFD检测系统。在优化的反应器膜长、毛细管内径及分离缓冲液和衍生溶液等条件下,对BSA的最低检测浓度可达到3.3nmol/L,并成功应用于实际蛋白样品的分析。
构建的LIFD具有调节简单,灵敏度高等特点,而LED-IFD具有结构通用性,可便捷地置换光源等组件,以完成不同的分析目的,为LIFD和LED-IFD的规模化生产和应用范围的拓宽奠定了基础。蛋白质柱后衍生膜反应器中的化学环境可以通过在衍生溶液中添加对应物质进行调节,同时可避免了样品的损失。通过选择不同种类的膜,可构建能够满足不同分析目的需要的膜反应器,并与HPLC\μHPLC以及CE系统联用,用于蛋白质、DNA等大分子的分离分析。
High separation efficiency and high detection sensitivity are the eternal goals of liquid chromatography. Benefit by the development of various kinds of advanced separation and detection methods, most samples can be well analyzed. But the analysis of ultra-low concentration materials needs further improvement of detection methods sensitivity. Fluorescence detection is sensitive and selective. Novel analysis systems with high separation efficiency and sensitivity can be built by coupling fluorescence detection with high performance liquid chromatography (HPLC) or capillary electrophoresis (CE), and the systems shall be effective tools for complex samples analysis.
Multiple fluorescence detectors were built using473nm laser,365nm and335nm light emitting diode (LED) as light source in this paper. Then a novel HPLC-LIFD system was constructed by coupling laser induced fluorescence detector (LIFD) with HPLC. Since a three-dimensional adjustable supportor of reflector and optimized structure were used in the LIFD, a10"12mol/L LOD of FITC was achieved when using the novel LIFD as detector. The trace bioamines in water were analyzed by the HPLC-LIFD system, and LODs of spermidine, putrescine and histamine were0.13、0.22and0.23nmol/L respectively, the recoveries were in the range of95%-105%. The HPLC-LIFD system was also used to analy fluorescent materials and amines in tea, about15～35fluorescent materials and more than48amines were detected in7teas, the type and content differences of the fluorescent materials and amines among the7teas were discovered.
Base on the advantage of shape emission profile and large stokes shift of Eu fluorescence probes, a HPLC-LED-IFD system special for Eu fluorescence probes was built, using a universal and modularized confocal optical system. The BSA, which was derivatized by BHHCT-Eu, was analyzed by the HPLC-LED-IFD system, and a10-8mol/L LOD was achieved. Then several materials derivatized by BHHCT-Eu were analyzed by the HPLC-LED-IFD system with different HPLC modes. It was confirmed that the BHHCT-Eu derivatized proteins might be analyzed by the reversed phase HPLC-LED-IFD system. Furthermore, base on the Eu fluorescence probes specialized LED-IFD, a365nm LED-IFD was built by changing the LED and optical filters, and the365nm LED-IFD might be used to detect benzopyrene.
Base on the macromolecules holding and small molecules exchange characteristic of the hollow fiber dialysis membrane, a novel post-column derivatization membrane reactor for protein analysis in CE has been designed. And a protein post-column derivatization membrane reactor based CE-LIFD system was constructed by combining the reactor, CE and LIFD. The membrane length, capillary inner diameter, separation buffer and derivatization solution were optimized. Under the optimized conditions, BSA was detected and a3.3nmol/L LOD was achieved. The system was furtherly successfully applied in the real protein samples analysis.
The LIFD built in this paper was sample and sensitive. The structure of the LED-IFD was universal, and other LED-IFD can be built for other samples analysis by changing the LED and filters. The foundation of the large scale production and broaden application range of LIFD and LED-IFD was strengthened by this paper. The chemical conditions in the post-column derivatization membrane reactor could be adjusted by the derivatization solution without sample loss, and other membrane reactors can be built using different membranes to satisfy the requirements of different samples analysis. The membrane reactor can be used in the HPLC, μHPLC or CE system for analysis of macromolecules, such as proteins and DNA.
采用473nm固体激光器和365nm、335nm发光二极管(LED)为光源,构建了多种HPLC用荧光检测器。将激光诱导检测器(LIFD)与HPLC结合构建成新型HPLC-LIFD系统。基于精密三维调节架的使用及光路优化设计,LIFD对FITC的最低检测浓度达到10-12mol/L。使用该系统分析水中微量生物胺,亚精胺、腐胺和组胺的检测限分别为0.13、0.22和0.23(?)nmol/L,回收率：95%-105%。进一步将该系统用于不同类型茶叶茶水中荧光物质及胺类物质的分析,共检测到15-35荧光物质和48种以上胺类物质,也揭示了不同茶叶茶水中荧光物质和胺类物质种类和含量上的差异。
基于铕离子(Eu)荧光探针的窄范围发射光谱和大斯托克斯位移优势,设计通用的模块化共聚焦光路系统,构建了铕离子荧光探针专用HPLC-LED-IFD分析系统。对Eu荧光探针BHHCT-Eu衍生的BSA进行分析,检测限达到10-8mol/L。考察了Eu荧光探针衍生化物质在不同模式HPLC中的分离特征,证实BHHCT-Eu衍生化蛋白质可在反相色谱模式下操作。通过置换LED光源和滤光片,也搭建了适用于苯并检测的365nm LED-IFD。
充分考虑中空纤维透析膜的截留大分子及小分子置换的特征,设计了一种新型高效毛细管电泳蛋白质柱后衍生膜反应器,并将之与CE系统和LIFD结合,构建了基于蛋白质柱后衍生膜反应器的CE-LIFD检测系统。在优化的反应器膜长、毛细管内径及分离缓冲液和衍生溶液等条件下,对BSA的最低检测浓度可达到3.3nmol/L,并成功应用于实际蛋白样品的分析。
构建的LIFD具有调节简单,灵敏度高等特点,而LED-IFD具有结构通用性,可便捷地置换光源等组件,以完成不同的分析目的,为LIFD和LED-IFD的规模化生产和应用范围的拓宽奠定了基础。蛋白质柱后衍生膜反应器中的化学环境可以通过在衍生溶液中添加对应物质进行调节,同时可避免了样品的损失。通过选择不同种类的膜,可构建能够满足不同分析目的需要的膜反应器,并与HPLC\μHPLC以及CE系统联用,用于蛋白质、DNA等大分子的分离分析。
High separation efficiency and high detection sensitivity are the eternal goals of liquid chromatography. Benefit by the development of various kinds of advanced separation and detection methods, most samples can be well analyzed. But the analysis of ultra-low concentration materials needs further improvement of detection methods sensitivity. Fluorescence detection is sensitive and selective. Novel analysis systems with high separation efficiency and sensitivity can be built by coupling fluorescence detection with high performance liquid chromatography (HPLC) or capillary electrophoresis (CE), and the systems shall be effective tools for complex samples analysis.
Multiple fluorescence detectors were built using473nm laser,365nm and335nm light emitting diode (LED) as light source in this paper. Then a novel HPLC-LIFD system was constructed by coupling laser induced fluorescence detector (LIFD) with HPLC. Since a three-dimensional adjustable supportor of reflector and optimized structure were used in the LIFD, a10"12mol/L LOD of FITC was achieved when using the novel LIFD as detector. The trace bioamines in water were analyzed by the HPLC-LIFD system, and LODs of spermidine, putrescine and histamine were0.13、0.22and0.23nmol/L respectively, the recoveries were in the range of95%-105%. The HPLC-LIFD system was also used to analy fluorescent materials and amines in tea, about15～35fluorescent materials and more than48amines were detected in7teas, the type and content differences of the fluorescent materials and amines among the7teas were discovered.
Base on the advantage of shape emission profile and large stokes shift of Eu fluorescence probes, a HPLC-LED-IFD system special for Eu fluorescence probes was built, using a universal and modularized confocal optical system. The BSA, which was derivatized by BHHCT-Eu, was analyzed by the HPLC-LED-IFD system, and a10-8mol/L LOD was achieved. Then several materials derivatized by BHHCT-Eu were analyzed by the HPLC-LED-IFD system with different HPLC modes. It was confirmed that the BHHCT-Eu derivatized proteins might be analyzed by the reversed phase HPLC-LED-IFD system. Furthermore, base on the Eu fluorescence probes specialized LED-IFD, a365nm LED-IFD was built by changing the LED and optical filters, and the365nm LED-IFD might be used to detect benzopyrene.
Base on the macromolecules holding and small molecules exchange characteristic of the hollow fiber dialysis membrane, a novel post-column derivatization membrane reactor for protein analysis in CE has been designed. And a protein post-column derivatization membrane reactor based CE-LIFD system was constructed by combining the reactor, CE and LIFD. The membrane length, capillary inner diameter, separation buffer and derivatization solution were optimized. Under the optimized conditions, BSA was detected and a3.3nmol/L LOD was achieved. The system was furtherly successfully applied in the real protein samples analysis.
The LIFD built in this paper was sample and sensitive. The structure of the LED-IFD was universal, and other LED-IFD can be built for other samples analysis by changing the LED and filters. The foundation of the large scale production and broaden application range of LIFD and LED-IFD was strengthened by this paper. The chemical conditions in the post-column derivatization membrane reactor could be adjusted by the derivatization solution without sample loss, and other membrane reactors can be built using different membranes to satisfy the requirements of different samples analysis. The membrane reactor can be used in the HPLC, μHPLC or CE system for analysis of macromolecules, such as proteins and DNA.
引文
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