Ru(II) encapsulated phosphorylate-terminated silica nanoparticles-based electrochemiluminescent strategy for label-free assay of protein kinase activity and inhibition.

A highly sensitive and simple label-free electrochemiluminescent (ECL) sensing strategy has been developed for assay of protein kinase A (PKA) activity and inhibition by taking advantage of zirconium cation (Zr(4+)) mediated signal transition and signal amplification of Ru(II) encapsulated phosphorylate-terminated silica nanoparticles (R-PSiNPs). In the protocol, an N-terminally cysteine-containing peptide (S-peptide) is self-assembled onto the gold electrode via Au-S bonding and used as substrate for PKA. The R-PSiNPs are chosen as the signal indicator by virtue of the intrinsic phosphate groups on the surface of the silica nanoparticles and the high loading of Ru(II) markers for ECL signal generation and amplification. The substrate peptide on the electrode is phosphorylated by PKA in the presence of ATP. The phosphorylated peptide (P-peptide) is subsequently linked with the R-PSiNPs by Zr(4+). The R-PSiNPs then can be grafted to the surface of Au electrode and generate high ECL signal. The ECL intensity is proportional to the activity of PKA. Due to the high loading of Ru(II) markers in a single phosphorylate-terminated silica nanoparticle, this strategy can be employed to assay PKA activity with a low detection limit of 0.005 U/mL. The linear range of the assay for PKA was 0.01 U/mL to 1 U/mL. Furthermore, the interferences experiments of CK2 and PKA inhibition have been also studied by using this strategy. This selective and sensitive method does not require labeling of the substrate peptide with ECL molecules, which provides a diversified platform for kinase activity and inhibition assay.