Highly sensitive electrogenerated chemiluminescence biosensor in profiling protein kinase activity and inhibition using a multifunctional nanoprobe

We presented a novel electrogenerated chemiluminescence (ECL) biosensor for monitoring the activity and inhibition of protein kinases based on signal amplification using enzyme-functionalized Au NPs nanoprobe. In this design, the biotin-DNA labeled glucose oxidase/Au NPs (GOx/Au NPs/DNA-biotin) nanoprobes, prepared by conjugating Au NPs with biotin-DNA and GOx, were bound to the biotinylated anti-phosphoserine labeled phosphorylated peptide modified electrode surface through a biotin−avidin interaction. The GOx assembled on the nanoprobe can catalyze glucose to generate H₂O₂ in the presence of O₂ while the ECL reaction occurred in the luminol ECL biosensor. At a higher concentration of kinase, there are more nanoprobes on the electrode, which gives a higher amount of GOx at the electrode interface and thus higher electrocatalytic efficiency to the luminol ECL reaction. Therefore, the activity of protein kinases can be monitored by ECL with high sensitivity. Protein kinase A (PKA), an important enzyme in regulation of glycogen, sugar, and lipid metabolism in the human body, was used as a model to confirm the present proof-of-concept strategy. The as-proposed biosensor presents high sensitivity, low detection limit of 0.013 U mL⁻¹, wide linear range (from 0.02 to 40 U mL⁻¹), and excellent stability. Moreover, this biosensor can also be used for quantitative analysis of kinase inhibition. On the basis of the inhibitor concentration dependent ECL signal, the half-maximal inhibition value IC₅₀ of ellagic acid, a typical PKA inhibitor, was estimated, which is in agreement with those obtained using the conventional kinase assay. The simple and sensitive biosensor is promising in developing a high-through assay of in vitro kinase activity and inhibitor screening for clinic diagnostic and drug development.