Protein kinases-mediated phosphorylation plays a crucial role in regulating cellular signal transduction and metabolic pathways, and aberrant kinase activity may induce multiple malignant diseases. Profiling of protein kinase activity is thus essential for understanding fundamental physiopathologic processes and for drug discovery.<br/>Some traditional bulk measurements for kinase activity measurement (such as mass spectrometry, fluorescence etc.) have been proposed in recent years and suffer from disadvantages such as requiring sophisticated equipment, involving time-consuming or labor-intensive labeling, and especially mediocre sensitivity. To overcome this problem, a series of signal amplification strategies have been introduced to improve the sensitivity. However, these approaches are capable of highly sensitive detection of kinase, nonspecific amplification may contribute to a false positive signal and compromise the assay accuracy. Consequently, amplification-free methods with enhanced sensitivity are highly desired for facile and accurate quantification of kinase activity.<br/>Here we develop a novel single-particle enumeration method for label-free and ultrasensitive probing of protein kinase A (PKA) activity. The PKA-mediated phosphorylation incorporates the biotin-conjugated phosphate entity into the substrate peptides, drives the assembly of streptavidin-functionalized gold nanoparticles, and generates intense scattering signals from the immobilized GNPs under dark-field illumination. In comparison to existing strategies, the proposed new method demonstrates five orders of linear range and improves the detection limit up to 10-to-1000 fold without the involvement of target amplification. Using this method, the PKA activity in MCF-7 cell lysates can be detected down to the quasi single cell levels. The method has also been successfully employed for quantitative kinase inhibitor screening and detection of chemical-induced PKA activity fluctuation in living cells, implying great potential in protein kinase-related biomedical, clinical, and pharmaceutical researches.

Phosphorylation-mediated single-particle assay of protein kinase activity with dark-field microscopy

2019 Fall National Meeting

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