Improved Laser-Induced Fluorescence Detection for Capillary Electrophoresis

将激光诱导荧光(LIF)检测引入分离科学，极大地提高了对各种分析物的检测灵敏度。通过开发吸收光谱与已知激光谱线匹配的荧光标记，这种类型的探测器的范围和应用得到了进一步的提高。虽然市面上有许多商用激光诱导荧光探测器，但实验室设计的、专门建造的系统可以提供更高的灵敏度和更多的功能。近年来，我们团队的研究人员开发了一种用于分离和生物科学应用的超灵敏LIF毛细管检测器，其P物质的灵敏度达到450fg/ml。 为了满足对内标的需求，我们团队在设计中引入了第二个波长功能，用于同时测量同一样品中的内标和未知分析物。内标用联苯双胺标记，人血浆样品用AlexaFluor633标记。在每个样品中加入含有每种标准品100 pg的混合物，然后注入毛细管电泳仪。样品在75 mA恒流下运行，用距进样口60 cm的流动池在线检测分解峰。荧光信号由408 nm二极管泵浦固体激光器和633 nm氦-霓激光共线组合而成的双色探测器测量，并在流动池处共聚焦。用一根光纤收集发射的光，光纤以90度角放置，并靠近流动池。准直光束通过417 nm长通拉曼边缘滤光器和633 nm激光陷波滤光器相结合。产生的信号通过第二根光纤传输到配备了数据采集板和LabVIEW接口的CCD光谱仪的入射狭缝。得到的两个色谱图被绘制成荧光单位与时间的关系图，并有堆叠的痕迹。 这种新的激光诱导荧光(LIF)检测器改进了我们以前的工作，允许同时检测用一个荧光团(Bimane)标记的内标和用不同荧光团标记的血清样本(AlexaFluor633)。这使研究人员能够对各种生物标本进行多分析物分析。通过计算峰面积并将其与标准物质进行直接比较来确定天然物质的定量。双色检测器的优点是，直接计算未知数可以缩短分析时间，并且不需要额外的标准或校准运行。双色激光诱导荧光检测的另一个优点是，在检测过程中可以获得高度的灵敏度，并且可以引入几个标准，从而允许多分析物的鉴定和定量。此外，同时检测同一样品中的标准物质和未知物，大大减少了分析时间。最后，减少的分析时间加上内置的质量控制使该方法成为临床研究和患者监测的理想方法。 今年，我们组装了一种新版本的双色检测器，优化了从50微米内径的方形毛细管中进行检测的能力。激光波长也被改变为660 nm和780 nm，以减少样品自发荧光的贡献。在研究了许多不同的配置后，我们选择了一个使用大数值孔径准直透镜的系统，以及一个针孔来排除不想要的背景。与早期设计的CCD探测器不同，收集的光被引导到光电倍增管上，每个波长对应一个光电倍增管。这些变化，加上新近可用的固态激光器的加入，已经导致检测器的总体尺寸显著减小，从而在在线系统中从分离到检测的距离大大减小。在这种配置中，检测器被优化为与纳米流高效液相色谱系统一起使用；将检测器合并到该系统中应该在本日历年末开始。

The introduction of laser-induced fluorescence (LIF) detection to separation science has greatly improved detection sensitivity for a variety of analytes. The range and application of this type of detector has been further enhanced by the development of fluorophore labels with absorption spectra matched to known laser lines. Although a number of commercial LIF detectors are available, laboratory-designed, purpose-built systems can offer improved sensitivity as well as added function. In recent years, researchers in our group developed an ultrasensitive LIF capillary detector for applications in the separation and biological sciences that achieved a sensitivity of 450 fg/ml of Substance P. To address the need for internal standards, our group introduced a second wavelength capability into the design for simultaneous measurement of internal standards and unknown analytes within the same sample. The internal standards were labeled with Bimane while human plasma samples were labeled with AlexaFluor633. Each sample was spiked with a mixture containing 100 pg of each standard and injected into a capillary electrophoresis system. The samples were run at 75 mA constant current and the resolved peaks detected on-line with a flow-cell set 60 cm from the inlet. The fluorescent signals were measured by the two-color detector, consisting of a 408-nm diode pumped solid state and a 633-nm helium-neon laser co-linearly combined and brought to common focus at the flow-cell. Emitted light was collected with an optical fiber, positioned at a 90-degree angle and in close proximity to the flow-cell. A collimated beam was passed through a 417-nm long pass Raman edge filter combined with a 633-nm laser notch filter. The resulting signal was transmitted via a second optical fiber to the entrance slit of a CCD spectrometer equipped with a data acquisition board and a Labview interface. The two resulting chromatograms were plotted as fluorescence units versus time with stacked traces. This new laser induced fluorescence (LIF) detector improves on our previous work by allowing the simultaneous detection of internal standards that are labeled with one fluorophore (Bimane) and serum samples labeled with a different fluorophore (AlexaFluor633). This enables investigators to perform multi-analyte analyses on a variety of biological specimens. Quantification of the natural materials is determined by calculating peak areas and directly comparing them with those obtained with the standards. The advantage of the two-color detector is that direct calculation of unknowns can shorten analytical time and negate the need for additional standard or calibration runs. An added advantage of two-color LIF detection is that a high degree of sensitivity can be achieved during detection and that several standards can be introduced, thus allowing for multi-analyte identification and quantification. Additionally, the simultaneous detection of standards and unknowns, within the same sample, greatly reduces analytical time. Finally, the reduced analytical time plus the in-built quality control makes this approach ideal for clinical studies and patient monitoring. This year, we have put together a new version of the two-color detector optimized for detection from a square capillary of 50 micron internal diameter. The laser wavelengths have also been changed to 660 nm and 780 nm to reduce the contribution from sample autofluorescence. After investigating a number of different configurations, we have chosen a system which uses high numerical aperture collimating lenses, together with a pinhole to exclude unwanted background. The collected light is directed onto photomultiplier tubes, one for each wavelength, in contrast to the CCD detector of the earlier design. These changes, together with the incorporation of newly available solid-state lasers, have resulted in a substantial decrease in the overall size of the detector, and hence the distance from the separation to the detection in the on-line system. In this configuration, the detector is optimized for use with a nanoflow HPLC system; incorporation of the detector into this system should begin by the end of the calendar year.