Miniature In-line Microfluidic LIF Detector

高压液相色谱（HPLC）和随后的质谱（MS）分析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MS入口的微流HPLC，进一步增强了这种方法。虽然这可以大大节省样品材料，但许多可用MS解析的峰低于标准或微型UV HPLC检测器的当前检测限。使用荧光检测，其具有显著更高的灵敏度，允许在这些微流和纳流系统中进行MS分析之前在线连续监测HPLC流出物。荧光检测与MS分析的结合允许对所研究的样品进行多维分析：使用内标物，可以在MS表征之前对每个解析峰进行定量。为了实现这一进步的MS技术，微型在线微流控激光诱导荧光（LIF）检测器的设计和建造。该检测器的关键特征是其便携性，其允许在HPLC分离之后和MS分析之前立即容易地插入毛细管微流体流动路径。这是通过将光纤用于激光诱导荧光检测器的激发和发射路径来实现的，其允许大体积光源、光学过滤元件和光子检测器远离检测点被容纳。 小型（4x 4x 1cm）原型探测器头由嵌入式通道组成，以容纳短长度的二氧化硅毛细管，具有385微米的外径和100微米直径的方形内部流动路径。通过去除约2 mm的聚酰亚胺涂层，将微米熔融石英光纤定位在毛细管中形成的观察窗附近。该光纤传导激发光（在这种情况下为405 nm）以诱导用萘二甲醛（NDA）标记的样品分析物的荧光。 第二光纤，600微米的熔融石英，也被定位在相同的观察点，并垂直于毛细管和激发纤维。由该光纤收集的光通过薄膜滤波器以消除散射的激光，并使用光电倍增管检测剩余的荧光信号。来自光电倍增管的信号可以被任何数字或模拟电压记录器读取。完整的检测器头，包括毛细管，互连和一个不透光的外壳，长度约为10厘米，横截面为5厘米。 观察样品体积约为6 nl，通过LIF与NDA肽标记的脱辅基肌红蛋白的胰蛋白酶消化物（用作蛋白质组学分析的模型样品）实现50 fmol或更低的灵敏度。色谱图显示在5微升/分钟的流速下具有出色的分辨率。实现的灵敏度和分辨率使该检测器处于当前最先进的水平，再加上其紧凑性和便携性，使其成为HPLC/MS技术的重大进步。未来的努力将包括纳入第二波长的激发和检测，特别是天然色氨酸荧光的检测。

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.