Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, Key Laboratory of Analytical, Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an 710062, Shaanxi Province (P.R. China).
Angew Chem Int Ed Engl. 2015 Dec 7;54(50):15186-90. doi: 10.1002/anie.201507580. Epub 2015 Oct 20.
A single microbead-based fluorescence imaging (SBFI) strategy that enables detection of protein kinase activity from single cell lysates is reported. We systematically investigated the ability of various rare earth (RE) ions, immobilized on the microbead, for specific capturing of kinase-induced phosphopeptides, and Dy(3+) was found to be the most prominent one. Through the efficient concentration of kinase-induced fluorescent phosphopeptides on a Dy(3+) -functionalized single microbead, kinase activity can be detected and quantified by reading the fluorescence on the microbead with a confocal fluorescence microscope. Owing to the extremely specific recognition of Dy(3+) towards phosphopeptides and the highly-concentrated fluorescence accumulation on only one microbead, ultrahigh sensitivity has been achieved for the SBFI strategy which allows direct kinase analysis at the single-cell level.
本文报道了一种基于单个微珠的荧光成像（SBFI）策略，可从单细胞裂解物中检测蛋白激酶活性。我们系统地研究了各种稀土（RE）离子在微珠上的固定化对激酶诱导的磷酸肽的特异性捕获能力，发现 Dy(3+) 是最突出的一种。通过在 Dy(3+)功能化的单个微珠上高效浓缩激酶诱导的荧光磷酸肽，可以通过共聚焦荧光显微镜读取微珠上的荧光来检测和定量激酶活性。由于 Dy(3+)对磷酸肽具有极其特异的识别能力，并且仅在一个微珠上高度浓缩荧光积累，因此 SBFI 策略实现了超高的灵敏度，可在单细胞水平上直接进行激酶分析。
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