Sulfur-Free Reversible Addition Fragmentation Chain Transfer Emulsion Polymerization Strategy for Electrochemical Analysis of Protein Kinase A Activity

In classical reversible addition fragmentation chain transfer (RAFT) polymerization, the use of sulfur-containing reagents is required, which inevitably causes some pollution to the environment. From the perspective of green chemistry, the use of nontoxic and harmless reactants does not pollute the environment and builds environmentally friendly biosensors. In this study, we proposed a sulfur-free RAFT electrochemical biosensor without a transition metal or sulfur catalyst using protein kinase A (PKA) as a model analyte. In this sensing process, a peptide consisting of a kinase-specific recognition sequence was designed. The peptide can be attached to the negatively charged surface of an APTMS-modified indium tin oxide (ITO) electrode (APTMS/ITO) via an amide reaction. When PKA was present, the substrate peptide was phosphorylated, which was then immediately coordinated with Zr4+. Then, Zr4+ was used as a linker to adsorb carboxyl-modified hollow gold nanoparticles (HAuNPs) to the electrode surface. Then, the SH-β-CD@2-NH2-Ada inclusion complex was conjugated to the HAuNPs via Au–S, and the electrode was immersed into the polymerization solution of catalytic chain transfer polymerization (CCTP) RAFT, resulting in an increased electrochemical signal. The sensing platform offers the advantages of simplicity, ease of handling, and enhanced phosphorylation, thanks to emulsion polymerization without the need for complex modification or immobilization processes. Also, the method is environmentally friendly, promises promising results, and represents a major advance in the development of complex macromolecular preparation. Basically, no toxic and dangerous chemical reagents are used in the entire biomolecular detection process. Moreover, given the pivotal role of cell lysate inhibition and the detection of protein kinase activity, this biosensor demonstrates promising prospects in kinase-related bioanalysis and clinical biomedicine.