Plasmonic AuNP/g‑C₃N₄ Nanohybrid-based Photoelectrochemical Sensing Platform for Ultrasensitive Monitoring of Polynucleotide Kinase Activity Accompanying DNAzyme-Catalyzed Precipitation Amplification

Abstract

A convenient and feasible photoelectrochemical (PEC) sensing platform based on gold nanoparticles-decorated g-C₃N₄ nanosheets (AuNP/g-C₃N₄) was designed for highly sensitive monitoring of T4 polynucleotide kinase (PNK) activity, using DNAzyme-mediated catalytic precipitation amplification. To realize our design, the AuNP/g-C₃N₄ nanohybrid was initially synthesized through in situ reduction of Au­(III) on the g-C₃N₄ nanosheets, which was utilized for the immobilization of hairpin DNA₁ (HP₁) on the sensing interface. Thereafter, a target-induced isothermal amplification was automatically carried out on hairpin DNA₂ (HP₂) in the solution phase through PNK-catalyzed 5′-phosphorylation accompanying formation of numerous trigger DNA fragments, which could induce generation of hemin/G-quadruplex-based DNAzyme on hairpin DNA₁. Subsequently, the DNAzyme could catalyze the 4-chloro-1-naphthol (4-CN) oxidation to produce an insoluble precipitation on the AuNP/g-C₃N₄ surface, thereby resulting in the local alternation of the photocurrent. Experimental results revealed that introduction of AuNP on the g-C₃N₄ could cause a ∼100% increase in the photocurrent because of surface plasmon resonance-enhanced light harvesting and separation of photogenerated e^–/h⁺ pairs. Under the optimal conditions, the percentage of photocurrent decrement (Δ<i>I</i>/<i>I</i>₀, relative to background signal) increased with the increasing PNK activity in a dynamic working range from 2 to 100 mU mL^–1 with a low detection limit (LOD) of 1.0 mU mL^–1. The inhibition effect of adenosine diphosphate also received a good performance in PNK inhibitor screening research, thereby providing a useful scheme for practical use in quantitative PNK activity assay for life science and biological research