Rare Earth Ion Mediated Fluorescence Accumulation on a Single Microbead: An Ultrasensitive Strategy for the Detection of Protein Kinase Activity at the Single-Cell Level.
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.
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).
Xiaobo
X
Zhang
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). liuch@snnu.edu.cn.
Chenghui
C
Liu
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).
Honghong
H
Wang
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).
Hui
H
Wang
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). lzpbd@snnu.edu.cn.
Zhengping
Z
Li
D004419:Dysprosium / Q000737:chemistry*; D004789:Enzyme Activation; D005453:Fluorescence; D006801:Humans; D007477:Ions / Q000737:chemistry; D061986:MCF-7 Cells; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D059010:Single-Cell Analysis
D007477:Ions; D004419:Dysprosium; D011494:Protein Kinases
fluorescence imaging; kinases; rare earths; single cells; single microbeads