Phosphopeptide-bridged NH2-TiO2-mediated carbon dots self-enhancing and electrochemiluminescence microsensors for label-free protein kinase A detection

A novel electrochemiluminescence (ECL) method was developed for analyzing protein kinase A (PKA) ultra-sensitively based on amidated nano-titanium (NH₂-TiO₂) embellished carbon dots (Mg@N-CDs) fluorescent probe, which integrated the target recognition and ECL-signal enhancement. The Cys-labeled kemptides were employed to build a serine-rich synthetic substrate-heptapeptide (Cys-kemptide) on the Au-electrode surface. Then, the PKA-induced biosensor was triggered as a signal switch to introduce the large amounts of TiO₂ decorated Mg@N-CDs nanohybrid (Ti@NMg-CDs) into AuE/Cys-phosphopeptides for signal output. In particular, the presence of PKA could induce the formation of Cys-phosphopeptides by the catalytic reaction between specific substrate (kemptide) and PKA, which could act as an initiator to link the Ti@NMg-CDs according to the bridge interactions Ti-O-P. By this way, multiple Cys-phosphopeptides were adsorbed onto a single Ti@NMg-CDs and the Ti@NMg-CDs not only provided the high specific selectivity but also large surface area, as well as unprecedented high ECL efficiency. Using this PKA-induced enhanced sensor, the limit-of-detection of the PKA was 4.89 × 10^{− 4} U/mL (S/N = 3). The proposed ECL biosensor was also universally applicable for the screening of PKA inhibitors and the determining of other kinases activity. Our sensing-system has excellent performance of specificity and the screening of kinase inhibitors, as well as it will inspire future effort on clinical diagnostics and new drugs discovery.