Institut für Funktionelle Grenzflächen, Karlsruhe Institute of Technology (KIT), Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany.
J R Soc Interface. 2011 Aug 7;8(61):1104-13. doi: 10.1098/rsif.2010.0594. Epub 2011 Jan 19.
We describe a method for multiplexed analysis of proteins using fluorescently encoded microbeads. The sensitivity of our method is comparable to the sensitivity obtained by enzyme-linked immunosorbent assay while only 5 µl sample volumes are needed. Streptavidin-coated, 1 µm beads are encoded with a combination of fluorophores at different intensity levels. As a proof of concept, we demonstrate that 27 microbead populations can be readily encoded by affinity conjugation using three intensity levels for each of three different biotinylated fluorescent dyes. Four populations of encoded microbeads are further conjugated with biotinylated capture antibodies and then combined and immobilized in a microfluidic flow cell for multiplexed protein analysis. Using four uniquely encoded microbead populations, we show that a cancer biomarker and three cytokine proteins can be analysed quantitatively in the picogram per millilitre range by fluorescence microscopy in a single assay. Our method will allow for the fabrication of high density, bead-based antibody arrays for multiplexed protein analysis using integrated microfluidic devices and automated sample processing.
我们描述了一种使用荧光编码微球进行蛋白质多重分析的方法。与酶联免疫吸附测定相比，我们的方法具有相同的灵敏度，而仅需 5µl 的样品量。链霉亲和素包被的 1µm 微球采用不同强度水平的荧光染料组合进行编码。作为概念验证，我们证明使用亲和素偶联，通过三种不同的生物素化荧光染料中的每一种使用三种强度水平，可轻松编码 27 种微球群体。进一步将 4 种编码微球与生物素化的捕获抗体偶联，然后组合并固定在微流控流动池内进行多重蛋白质分析。使用 4 种独特编码的微球群体，我们通过荧光显微镜在单次测定中显示可以在 picogram 每毫升范围内定量分析癌症生物标志物和三种细胞因子蛋白。我们的方法将允许使用集成微流控装置和自动化样品处理，制造用于多重蛋白质分析的高密度、基于微球的抗体阵列。
