Self-powered detection of T4 polynucleotide kinase activity based on DNA structure transformation-modulated direct electron transfer of bilirubin oxidase.

Abstract

Enzymatic biofuel cell-based self-powered sensors represent a promising class of portable sensing devices, so the development of a novel and efficient self-powered sensing strategy is of critical importance. Herein, the direct electron transfer (DET) of bilirubin oxidase (BOD) is modulated through enzymatic reaction-triggered DNA structure transformation, which is further applied for self-powered detection of T4 polynucleotide kinase (T4 PNK) activity. The biocathode of a glucose/oxygen biofuel cell is prepared by immobilizing BOD on carbon nanotubes (MWCNTs)-gold nanoparticles (AuNPs) nanocomposite by using a short-stranded DNA (sDNA)-complementary DNA (cDNA) duplex as a bridge. For detecting T4 PNK activity, the biocathode is incubated with T4 PNK and adenosine triphosphate to phosphorylate the 5'-hydroxyl termini of sDNA, followed by λ-exonuclease digestion of the phosphorylated sDNA, resulting in structure transformation of cDNA into a hairpin configuration via intramolecular base-pairing. As a result, BOD is repositioned near the MWCNTs-AuNPs interface, permitting efficient oxygen reduction catalysis through the DET. Consequently, the glucose/oxygen biofuel cell switches from an initial "open-circuit" to a "closed-circuit" operational mode, enabling self-powered detection of T4 PNK activity. The linear range for T4 PNK activity detection is from 0.001 to 2 U mL -1 , with a low detection limit of 3 × 10 -4 U mL -1 . The self-powered sensor is successfully used for detcting T4 PNK activity in a human serum sample. This work presents a novel strategy for self-powered sensing by modulating the DET of BOD via enzymatic reaction-triggered DNA structure transformation.

Keywords

Identifiers

DOI

10.1016/j.talanta.2025.128686

PubMed

40782738

Journal

Talanta

Year

2025