Protein-protein interaction analysis in single microfluidic droplets using FRET and fluorescence lifetime detection
Protein-protein interaction analysis in single microfluidic droplets using FRET and fluorescence lifetime detection
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DOI：
10.1039/c3lc00057e
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发表时间：
2013-01-01
期刊：
影响因子：
6.1
通讯作者：
Belder, Detlev
中科院分区：
文献类型：
--
作者：
Benz, Christian;Retzbach, Heiko;Belder, Detlev
在此，我们证明了使用 FRET 和显微荧光寿命测量对微流体液滴进行蛋白质-蛋白质相互作用分析和反应进度监测的可行性。演示了使用软光刻技术制造微滴芯片，所得芯片在 7.9 和 0.75 Hz 的频率下分别可靠地生成 630 pL 和 6.71 nL 的微滴。它们用于使用 Alexa Fluor 488 标记的生物素化 BSA、Alexa Fluor 594 标记的链霉亲和素和未标记的鸡蛋清亲和素的模型系统检测微滴中的蛋白质-蛋白质相互作用。这些微芯片可用于以纳摩尔检测限（对应于阿托摩尔蛋白质量）的直接和竞争测定形式定量检测微滴中的抗生物素蛋白和链霉抗生物素蛋白。发现四个液滴足以进行分析测定。进行荧光强度比和荧光寿命测量并比较微滴 FRET 测定。使用荧光寿命检测测定未标记亲和素的竞争性芯片上结合测定只需在 135 秒内完成。
Herein, we demonstrate the feasibility of a protein-protein interaction analysis and reaction progress monitoring in microfluidic droplets using FRET and microscopic fluorescence lifetime measurements. The fabrication of microdroplet chips using soft-and photolithographic techniques is demonstrated and the resulting chips reliably generate microdroplets of 630 pL and 6.71 nL at frequencies of 7.9 and 0.75 Hz, respectively. They were used for detection of protein-protein interactions in microdroplets using a model system of Alexa Fluor 488 labelled biotinylated BSA, Alexa Fluor 594 labelled streptavidin and unlabelled chicken egg white avidin. These microchips could be used for quantitative detection of avidin and streptavidin in microdroplets in direct and competitive assay formats with nanomolar detection limits, corresponding to attomole protein amounts. Four droplets were found to be sufficient for analytical determination. Fluorescence intensity ratio and fluorescence lifetime measurements were performed and compared for microdroplet FRET determination. A competitive on-chip binding assay for determination of unlabelled avidin using fluorescence lifetime detection could be performed within 135 s only.