λ-exonuclease-driven split G-quadruplex hybridization-based DNA walking system for sensitive microRNA quantification

The precise measurement of microRNAs (miRNAs) is essential for diagnosing newborn pneumonia. This paper presents a simple, sensitive and accurate fluorescence-based technique for miRNA identification, utilizing a λ-exonuclease (λ-Exo)-driven DNA walker and split G-quadruplex (split-G4)-facilitated signal amplification. In this biosensor, target miRNA initiates the DNA walker by unfolding the Walker-probe, hence perpetually facilitating the reassembly of split-G4. The reformed intact G4 structure is distinctly identified by the commercially accessible fluorescent dye thioflavin T (ThT), facilitating highly sensitive, label-free miRNA identification. Additionally, the DNA walking process is motivated by the λ-Exo, which endows the biosensor with a greatly elevated signal amplification efficiency. This method demonstrates low background noise and good dependability owing to its reliance on split-G4-generated signals. Furthermore, the technique has been effectively utilized on clinical specimens, indicating its capability for disease diagnosis.