Improved Laser-Induced Fluorescence Detection for CE

临床和基础生物学研究样品中多分析物的检测和测量是几年来研究的主题。这导致除了减少分析时间外，还需要测量同一样品中的多个分析物。样品的比对？S未知的样品与标准样品的比对非常耗时，而且经常导致不一致，特别是在毛细管电泳法(CE)等专业技术中。在临床领域，对及时分析的需要是非常必要的，同时检测几个内部标准以及感兴趣的分析物的能力具有巨大的潜力。 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，DBEPS仪器研究与开发资源(IRDR)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。 为了满足内部标准的需求，IRDR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部标准和未知分析物。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，在检测过程中可以实现高度的灵敏度，并且可以引入几个标准，从而允许多分析物的识别和定量。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。

The detection and measurement of multi-analytes in clinical and basic biological research samples has been a subject of investigation for several years. This has lead to a growing need to measure multiple analytes in the same sample in addition to reducing analytical time. Comparison of the sample?s unknowns to standards is time-consuming and often leads to inconsistencies, especially in specialized techniques such as capillary electrophoresis (CE). In the clinical arena, the need for timely analyses is of great necessity and the ability to simultaneously detect several internal standards along with the analytes of interest holds great potential. 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, the DBEPS Instrumentation Research and Development Resource (IRDR) 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, IRDR 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 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.