Miniature In-line Microfluidic LIF Detector

高压液相色谱（HPLC）和随后的质谱分析（MS）是蛋白质组学领域重要的金标准技术。通过引入直接耦合到纳米喷雾质谱入口的微流HPLC，这种方法得到了进一步的增强。虽然这可以大大节省样品材料，但许多用MS可解析的峰低于标准或微型紫外高效液相色谱检测器的当前检测极限。荧光检测具有更高的灵敏度，允许在线连续监测HPLC流出物，然后在这些微和纳米流系统中进行质谱分析。荧光检测与质谱分析的耦合允许对所调查的样品进行多维分析：使用内部标准，可以在质谱表征之前对每个解析峰进行量化。为了实现质谱技术的这一进步，设计并构建了微型在线微流控激光诱导荧光（LIF）检测器。该检测器的主要特点是便携性，便于在HPLC分离后和质谱分析之前插入毛细管微流体流动路径。这是通过在激光诱导荧光探测器的激发和发射路径上使用光纤来实现的，这使得笨重的光源、光学过滤元件和光子探测器可以被安置在远离检测点的地方。

High-pressure liquid chromatography (HPLC) and subsequent analysis by mass spectrometry (MS) are important, gold-standard techniques in the field of proteomics. This approach has been further enhanced by the introduction of microflow HPLC coupled directly to nanospray MS inlets. Although this can greatly save on sample materials, many peaks that are resolvable with MS are below the current limit of detection for standard or micro UV HPLC detectors. The use of fluorescence detection, which has substantially greater sensitivity, allows on-line, continuous monitoring of the HPLC effluent prior to MS analysis in these micro and nanoflow systems. The coupling of fluorescence detection with MS analysis then allows for multi-dimensional analysis of the sample under investigation: with internal standards, each resolved peak can be quantified prior to MS characterization. To accomplish this advance in MS technology, a miniature in-line microfluidic laser induced fluorescence (LIF) detector was designed and constructed. The key feature of this detector is its portability, which permits easy insertion into the capillary microfluidic flow path after HPLC separation and immediately prior to MS analysis. This is enabled by the use of fiber-optics for both the excitation and emission paths of the laser induced fluorescence detector that allows bulky light sources, optical filtration elements, and photon detectors to be housed remotely from the point of detection. The small (4x4x1cm) prototype detector head consists of an embedded channel to accommodate a short length of silica capillary, with a 385-micron outside diameter and a square 100-micron diameter internal flow path. Perpendicular to the capillary is a second embedded channel to accommodate a 200-micron fused silica optical fiber that is positioned in close proximity to an observation window formed in the capillary by removal of approximately 2 mm of the polyimide coating. This fiber conducts excitation light (405 nm in this instance) to induce fluorescence of the sample analytes that are tagged with naphthalene dicarboxaldehyde (NDA). A second optical fiber, 600-micron fused silica, is also positioned at the same observation point and is perpendicular to both the capillary and the excitation fiber. The light collected by this optical fiber is passed through a thin-film filter to eliminate scattered laser light, and the remaining fluorescent signal is detected using a photomultiplier tube. The signal from the photomultiplier tube can be read by any voltage recorder, digital or analog. The complete detector head, including capillary tubing, interconnects and a light-tight housing is approximately 10 cm in length and 5 cm in cross section. The observation sample volume is approximately 6 nl and a sensitivity of 50 fmol or less was achieved by LIF with NDA peptide labelling of a tryptic digest of apomyoglobin that was used as a model sample for proteomic analysis. Chromatograms show excellent resolution at flow rates of 5 microliters/min. The sensitivity and resolution achieved place this detector at the current state-of the-art, which together with its compactness and portability make it a significant advance in HPLC/MS technology. Future efforts will include incorporating a second wavelength for excitation and detection, in particular the detection of native tryptophan fluorescence.