Ouellet Eric, Lund Louise, Lagally Eric T
Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada.
Methods Mol Biol. 2013;949:473-90. doi: 10.1007/978-1-62703-134-9_30.
The reliable detection of ligand and analyte binding is of significant importance for the field of medical diagnostics. Recent advances in proteomics and the rapid expansion in the number of identified protein biomarkers enhance the need for reliable techniques for their identification in complex samples. Surface plasmon resonance imaging (SPRi) provides label-free detection of this binding process in real-time. This chapter details the fabrication of an SPR imaging instrument and its use in analyzing molecular binding interactions with the use of a high-density microfluidic SPRi chip, capable of multiplexed analysis as well as various immobilization chemistries. Controlled recovery of bound biomarkers is demonstrated to enable their identification using mass spectrometry. Finally, activated leukocyte cell adhesion molecule (ALCAM), a protein biomarker associated with a variety of cancers, is identified from human crude cell lysates using the microfluidic surface plasmon resonance imaging (SPRi) instrument.
配体与分析物结合的可靠检测对于医学诊断领域至关重要。蛋白质组学的最新进展以及已鉴定蛋白质生物标志物数量的迅速增加，使得在复杂样品中鉴定它们的可靠技术的需求也日益增强。表面等离子体共振成像（SPRi）可实时对这种结合过程进行无标记检测。本章详细介绍了一台SPR成像仪器的制造及其在分析分子结合相互作用中的应用，该仪器使用了一种高密度微流控SPRi芯片，能够进行多重分析以及采用各种固定化学方法。结果表明，结合的生物标志物的可控回收能够使用质谱对其进行鉴定。最后，使用微流控表面等离子体共振成像（SPRi）仪器从人粗细胞裂解物中鉴定出了与多种癌症相关的蛋白质生物标志物——活化白细胞细胞黏附分子（ALCAM）。
