Improved Laser-Induced Fluorescence Detection for CE

改进的 CE 激光诱导荧光检测

• 批准号: 7146086
• 负责人: Nicole Y Morgan
• 金额: --
• 依托单位: OFFICE OF THE DIRECTOR, NATIONAL INSTITUTES OF HEALTH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7146086
• 关键词: capillary electrophoresis; fluorescent dye /probe; human tissue; ionophores; substance P; technology /technique development

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.

临床和基础生物学研究样品中的多分析物的检测和测量已经是多年的研究主题。这导致除了减少分析时间之外，还需要测量同一样品中的多种分析物。样品比较？对未知的标准品进行分析是一项耗时的工作，而且经常会导致不一致，特别是在毛细管电泳（CE）等专业技术中。在临床竞技场中，对及时分析的需求是非常必要的，并且同时检测几种内标物沿着感兴趣的分析物的能力具有很大的潜力。 将激光诱导荧光（LIF）检测引入分离科学，极大地提高了各种分析物的检测灵敏度。这种类型的检测器的范围和应用已被进一步增强的吸收光谱与已知的激光线相匹配的荧光标记的发展。虽然有许多商业LIF探测器可用，但实验室设计的专用系统可以提供更高的灵敏度和更多的功能。近年来，DBEPS仪器研究和开发资源（IRDR）开发了一种用于分离和生物科学的超灵敏LIF毛细管检测器，其对P物质的灵敏度达到450 fg/ml。 为了满足对内标物的需求，IRDR在设计中引入了第二波长功能，用于同时测量同一样品中的内标物和未知分析物。内标物用Bimane标记，而人血浆样品用AlexaFluor 633标记。每份样品加标含有100 pg各标准品的混合物，并进样至毛细管电泳系统中。样品在75 mA恒定电流下运行，并使用距入口60 cm的流动池组在线检测解析峰。通过双色检测器测量荧光信号，双色检测器由共线性组合的408 nm二极管泵浦固态和633 nm氦氖激光组成，并在流动池处共聚焦。发射的光用光纤收集，以90度角定位并紧邻流动池。准直光束通过与633 nm激光陷波滤波器组合的417 nm长通拉曼边缘滤波器。所得信号通过第二光纤传输到配备有数据采集板和Labview接口的CCD光谱仪的入口狭缝。将两个所得色谱图绘制为荧光单位与时间的关系图，其中具有堆叠迹线。 这种新的LIF检测器改进了我们以前的工作，允许同时检测用一种荧光团（Bimane）标记的内标物和用不同荧光团（AlexaFluor 633）标记的血清样品。这使研究人员能够对各种生物标本进行多分析物分析。通过计算峰面积并直接将其与标准品所得峰面积进行比较来确定天然材料的定量。双色检测器的优点是，直接计算未知量可以缩短分析时间，无需额外的标准或校准运行。双色LIF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，并且可以引入几种标准，从而允许多分析物鉴定和定量。此外，在同一样品中同时检测标准品和未知物，大大缩短了分析时间。最后，缩短的分析时间加上内置的质量控制使这种方法成为临床研究和患者监测的理想选择。