TiO<sub>2</sub>/g‑C<sub>3</sub>N<sub>4</sub>/CdS Nanocomposite-Based Photoelectrochemical Biosensor for Ultrasensitive\nEvaluation of T4 Polynucleotide Kinase Activity

Herein, an efficient\nphotoelectrochemical (PEC) platform was constructed\nby a cosensitization strategy with a cascade energy level arrangement\nfor the ultrasensitive evaluation of T4 polynucleotide kinase (T4\nPNK). Based on CdSe quantum dots (QDs) with an extremely narrow bandgap,\nthis cosensitization strategy offered a highly efficient sensitizer\nwith a matching band-edge level of a ternary TiO₂/g-C₃N₄/CdS nanocomposite. In this protocol, the ternary\nnanocomposite was first prepared to serve as the matrix to construct\nthe PEC sensing platform. On the other hand, a well-designed hairpin\nDNA₁ probe with 5′-hydroxyl termini was specifically\nphosphorylated by T4 PNK which would be selectively cleaved with lambda\nexonuclease (λ-Exo) outputting the 3′-thiol end ssDNA₂. After tagged with CdSe QDs, ssDNA₂ was captured\nby the complementary capture DNA₃ on the electrode surface.\nAs a result, CdSe QDs were in close contact with the ternary nanocomposite\nmatrix, leading to an enhanced photocurrent response. Therefore, this\nproposed PEC platform displayed an analytical performance with a wide\nlinear range from 0.0001 to 0.02 U mL^–1 and a low\ndetection limit down to 6.9 × 10^–5 U mL^–1. Moreover, this ternary nanocomposite-based platform\nexhibited excellent selectivity, good reproducibility, and remarkable\nstorage stability, which shows great potential for T4 PNK detection\nand inhibitor screening.