BioMolecular Analysis, Department of Chemistry and Pharmaceutical Sciences, Faculty of Sciences, VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
Anal Bioanal Chem. 2010 Dec;398(7-8):3023-32. doi: 10.1007/s00216-010-4210-x. Epub 2010 Sep 25.
One way to profile complex mixtures for receptor affinity is to couple liquid chromatography (LC) on-line to biochemical detection (BCD). A drawback of this hyphenated screening approach is the relatively high consumption of sample, receptor protein and (fluorescently labeled) tracer ligand. Here, we worked toward minimization of sample and reagent consumption, by coupling nano-LC on-line to a light-emitting diode (LED) based capillary confocal fluorescence detection system capable of on-line BCD with low-flow rates. In this fluorescence detection system, a capillary with an extended light path (bubble cell) was used as a detection cell in order to enhance sensitivity. The technology was applied to a fluorescent enhancement bioassay for the acetylcholine binding protein, a structural analog of the extracellular ligand-binding domain of neuronal nicotinic acetylcholine receptors. In the miniaturized setup, the sensitive and low void volume LED-induced confocal fluorescence detection system operated in flow injection analysis mode allowing the measurement of IC(50) values, which were comparable with those measured by a conventional plate reader bioassay. The current setup uses 50 nL as injection volume with a carrier flow rate of 400 nL/min. Finally, coupling of the detection system to gradient reversed-phase nano-LC allowed analysis of mixtures in order to identify the bioactive compounds present by injecting 10 nL of each mixture.
一种对复杂混合物进行受体亲和力分析的方法是将液相色谱（LC）与生化检测（BCD）在线耦合。这种联用筛选方法的一个缺点是样品、受体蛋白和（荧光标记的）示踪配体的相对高消耗量。在这里，我们通过将纳升液相色谱（nano-LC）在线耦合到能够以低流速在线进行 BCD 的基于发光二极管（LED）的毛细管共焦荧光检测系统，致力于减少样品和试剂的消耗。在这个荧光检测系统中，使用具有扩展光程的毛细管（气泡池）作为检测池，以提高灵敏度。该技术应用于乙酰胆碱结合蛋白的荧光增强生物测定，乙酰胆碱结合蛋白是神经元烟碱型乙酰胆碱受体细胞外配体结合域的结构类似物。在微型化设置中，灵敏且低空隙体积的 LED 诱导共焦荧光检测系统以流动注射分析模式运行，允许测量 IC50 值，这些值与通过传统平板读取器生物测定测量的值相当。当前设置使用 50 nL 的进样体积和 400 nL/min 的载气流速。最后，将检测系统与梯度反相纳升液相色谱耦合，允许分析混合物，通过注射每种混合物的 10 nL 来鉴定存在的生物活性化合物。
