Label-free fluorescent detection of protein kinase activity based on the aggregation behavior of unmodified quantum dots.
Herein, we present a novel label-free fluorescent assay for monitoring the activity and inhibition of protein kinases based on the aggregation behavior of unmodified CdTe quantum dots (QDs). In this assay, cationic substrate peptides induce the selective aggregation of unmodified QDs with anionic surface charge, whereas phosphorylated peptides do not. Phosphorylation by kinase alters the net charge of peptides and subsequently inhibits the aggregation of unmodified QDs, causing an enhanced fluorescence with a 45 nm blue-shift in emission and a yellow-to-green emission color change. Hence the fluorescence response allows this QD-based method to easily probe kinase activity by a spectrometer or even by the naked eye. The feasibility of the method has been demonstrated by sensitive measurement of the activity of cAMP-dependent protein kinase (PKA) with a low detection limit (0.47 mU μL(-1)). On the basis of the fluorescence response of QDs on the concentration of PKA inhibitor H-89, the IC(50) value, the half maximal inhibitory concentration, was estimated, which was in agreement with the literature value. Moreover, the system can be applicable to detect the Forskolin/3-isobutyl-1-methylxantine (IBMX)-stimulated activation of PKA in cell lysate. Unlike the existing QD-based enzyme activity assays in which the modification process of QDs is essential, this method relies on unmodified QDs without the requirement of peptide labeling and QDs' modification, presenting a promising candidate for cost-effective kinase activity and inhibitor screening assays.
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, PR China.
Xiahong
X
Xu
Xin
X
Liu
Zhou
Z
Nie
Yuliang
Y
Pan
Manli
M
Guo
Shouzhuo
S
Yao
D000595:Amino Acid Sequence; D019187:Cadmium Compounds / Q000737:chemistry; D002457:Cell Extracts; D045744:Cell Line, Tumor; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D057075:Enzyme Assays / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism*; D045663:Quantum Dots; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013499:Surface Properties; D013691:Tellurium / Q000737:chemistry
D019187:Cadmium Compounds; D002457:Cell Extracts; D005456:Fluorescent Dyes; D010455:Peptides; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D013691:Tellurium; C028337:cadmium telluride