Improved Laser-Induced Fluorescence Detection for Capillary Electrophoresis

改进的毛细管电泳激光诱导荧光检测

• 批准号: 7593834
• 负责人: Paul Smith
• 金额: $ 2.48万
• 依托单位: NATIONAL INSTITUTE OF BIOMEDICAL IMAGING AND BIOENGINEERING
• 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/7593834
• 关键词: Address; Area; Biological; Biological Sciences; Blood capillaries; Calendar; Caliber; Calibration; Capillary Electrophoresis; Cells; Clinical; Clinical Research; Color; Detection; Development; Fluorescence; Helium; High Pressure Liquid Chromatography; Human; Investigation; Label; Laboratories; Lasers; Lead; Light; Measurement; Measures; Neon; Numbers; Online Systems; Patient Monitoring; Plasma; Positioning Attribute; Pump; Purpose; Quality Control; Range; Research Personnel; Running; Sampling; Science; Serum; Signal Transduction; Specimen; Standards of Weights and Measures; Substance P; System; Techniques; Time; Tube; Work; absorption; biological research; capillary; data acquisition; design; detector; fluorophore; improved; interest; lens; notch protein; optical fiber; photomultiplier; size; solid state

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.

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，我们小组的研究人员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，我们的团队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。 这种新型激光诱导荧光（LIF）检测器改进了我们之前的工作，允许同时检测标记有一种荧光团（Bimane）的内标物和标记有不同荧光团（AlexaFluor 633）的血清样本。 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品，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。 今年，我们推出了一款新版本的双色检测器，该检测器针对内径为50微米的方形毛细管进行了优化。 激光波长也已变更为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，我们选择了一个系统，它使用高数值孔径准直透镜，连同针孔，以排除不需要的背景。 收集的光被引导到光电倍增管上，每个波长一个，与早期设计的CCD检测器形成对比。 这些变化，加上新的可用的固态激光器的结合，导致在检测器的整体尺寸，并因此从分离到在线系统中的检测的距离大幅减少。 在这种配置中，检测器被优化为与纳米流HPLC系统一起使用;将检测器并入该系统应在日历年年底前开始。