Biosensor development for diabetes diagnosis: Determining relevant miRNA using a newly developed N-annulated perylene fluorescent dye.

MicroRNAs (miRNAs) have emerged as essential biomarkers for disease diagnosis, and several techniques are available to determine type 2 diabetes (T2D) relevant miRNAs. However, detecting circulating miRNAs can be challenging due to their small size, low abundance, and high sequence similarity, often requiring sensitive detection approaches combined with additional amplification processes. Laser-induced fluorescence (LIF) is a classic analytical method suitable for sensitively detecting trace amounts of nucleotide acid. Duplex-specific nuclease (DSN)-mediated amplification recently gained attention due to its catalytic activity based on target recycling, demonstrating a promising approach for miRNA amplification. This work developed a novel N-annulated perylene fluorescent dye to create a biosensor to analyze the miRNA (miR-223) relevant to T2D. The amine-reactive fluorescent dye assists the amidation reaction for nucleotide labeling, giving the oligonucleotide probe a high fluorescence quantum yield and sufficient water solubility. By combining the locked nucleic acid (LNA) modified oligonucleotide fluorescent probe to enhance the stability of LNA/RNA hybrids, thereby improving the DSN-mediated target miR-223 recycling for signal amplification, the proposed biosensor can highly selectively determine miR-223 with a limit of detection (LOD, S/N = 3) of 9.5 pM. When applied to real-world samples, the biosensor demonstrated its potential to distinguish between T2D patients and healthy controls.