Split malachite green aptamer transcription-based AND logic fluorescent platform: A novel approach for dual-miRNA detection in diabetes mellitus complicated with Alzheimer's disease

An AND logic fluorescent chip is developed for dual-miRNA detection in diabetes mellitus with Alzheimer's disease. • Self-primer cyclic amplification and dual rolling circle amplification enable ultrasensitive miRNA analysis. • Split malachite green aptamer transcription allows label-free, catalytically amplified fluorescence signal generation. • The chip achieves 0.15 fM LOD for both miRNAs and excellent specificity against mismatched sequences. • Clinical serum analysis shows 87.5% sensitivity for DMAD diagnosis, outperforming single-miRNA RT-qPCR. Early diagnosis of Diabetes Mellitus complicated with Alzheimer's Disease (DMAD) remains a major clinical challenge, and reliable detection of multiple microRNA (miRNA) biomarkers holds great promise for improving diagnostic accuracy. Herein, we report a split malachite green (MG) aptamer transcription-based fluorescent platform that operates as an “AND” logic gate for simultaneous ultrasensitive detection of two DMAD-associated miRNAs, miRNA-34a and miRNA-132. The platform integrates self-primer cyclic amplification with surface-anchored dual rolling circle transcription (RCT). Only when both target miRNAs are present, a target-recycling process releases Y-shaped probes, which subsequently initiate dual RCT to produce long DNA products containing tandem repeats of split MG aptamer sequences. These two products assemble into an intact MG aptamer, catalytically generating a strong fluorescence signal. The assay achieves a limit of detection as low as 0.15 fM for both miRNAs, with excellent specificity against mismatched and non-target sequences. The platform exhibits high stability (recoveries of 94.1–99.5% in spiked serum) and good reproducibility (coefficients of variation of 4.12% and 5.32% at 100 fM and 10 pM, respectively). In 30 clinical serum samples, the method correlates strongly with RT-qPCR and distinguishes DMAD patients from healthy controls with 87.5% sensitivity, significantly outperforming single-miRNA RT-qPCR (62.5% sensitivity). This “AND” logic-based split aptamer transcription platform provides a sensitive, specific, robust, and user-friendly tool for multiplex miRNA analysis, offering great potential for early DMAD diagnosis and broader applications in complex diseases. Download: Download high-res image (94KB) Download: Download full-size image