Improved Laser-Induced Fluorescence Detection for Capill

改进的毛细管激光诱导荧光检测

• 批准号: 7319259
• 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/7319259
• 关键词: Improved; Laser; Induced; Fluorescence; Detection

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. This year, we have begun systematic studies of different configurations for the collecting optics and photodetectors for an improved version of the two-color detector. The hope is to improve upon sensitivity by using high numerical aperture collimating lenses, together with a pinhole to exclude unwanted background, and directing the collected light onto photomultiplier tubes, in contrast to the CCD detector of the earlier design. In addition, this configuration should permit the use of smaller-diameter capillary, allowing detection on systems with lower flow rates. These changes, together with the incorporation of newly available solid-state lasers, should also allow 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 the next year, we plan to incorporate this detector into a microflow HPLC system in DBEPS's Ultramicro Analytical Immunochemistry Resource.

多年来，临床和基础生物学研究样品中多种分析物的检测和测量一直是一个研究课题。除了减少分析时间外，这导致越来越需要在同一样品中测量多种分析物。将未知样品与标准样品进行比较非常耗时，而且常常导致不一致，特别是在毛细管电泳（CE）等专业技术中。在临床领域，对及时分析的需求是非常必要的，同时检测多个内部标准以及感兴趣的分析物的能力具有巨大的潜力。