Development of a phos-tag-based fluorescent biosensor for sensitive detection of protein kinase in cancer cells

Protein kinase can catalyze the phosphorylation of peptides/proteins, and it is closely associated with various human diseases such as cancer, immune deficiencies, and Alzheimer's disease. Sensitive monitoring of protein kinase activity is significant for biochemical research and drug discovery. Herein, we develop a phos-tag-based fluorescent biosensor for sensitive detection of cAMP-dependent protein kinase (PKA) activity in cancer cells. We design a peptide-DNA conjugate and a signal probe for PKA activity assay, and employ a biotinylated phos-tag (a selective phosphate-binding agent) to recognize and capture the phosphorylated peptide-DNA substrate. The peptide-DNA conjugates as the catalytic substrates can translate the peptide signal to a nucleotide signal for the initiation of the RNase HII-driven cycling signal amplification. The magnetic nanobeads (MBs) as the carriers can separate and enrich the phosphorylated peptide in complex matrices. Meanwhile, the combination of MBs with the phos-tag-mediated phosphate group recognition can effectively eliminate the interference from the complex matrix, and the introduction of single-molecule detection endows this assay with high sensitivity. This biosensor can achieve a detection limit of 1.98 × 10^-8 U μL^-1 and a wide dynamic range from 1 × 10^-7 to 1 × 10^-2 U μL^-1. Moreover, this biosensor can be applied for the screening of PKA inhibitor and the measurement of cellular PKA activity, holding great potential in biomedical research and clinical diagnosis.