Quantum dot-enhanced tunable FRET nanosensor for Alzheimer's-associated dual miR-34 detection

The microRNAs miR-34a and miR-34b are dysregulated in Alzheimer's disease (AD) and are promising diagnostic biomarkers. However, their low abundance, high sequence homology, and instability present severe challenges for accurate multiplex detection. Herein, we report a quantum dot-enhanced turn-up/down dual-miRNA FRET sensor (QDTF sensor), a platform that achieves simultaneous detection of two highly homologous miRNAs with low crosstalk via an unprecedented asymmetric dual-mode FRET strategy enhanceded by a single quantum dot 605. Among the two pairs of molecular beacons, one triggers a considerable increase in the Alexa Fluor 568 signal for miR-34a, while the other induces a decrease in the Alexa Fluor 532/Cy5 signal for miR-34b. The deliberate opposite-signaling design and spectrally well-separated dye pairs present excellent channel isolation with low crosstalk (<3.42%), even when both targets are present at high concentrations. The QDTF sensor delivers LODs of 0.96 pM (miR-34a) and 1.12 pM (miR-34b) directly in artificial cerebrospinal fluid (aCSF) without enzymes, amplification, or pretreatment, with a dynamic range spanning approximately 1 pM to 10 nM, representing a 322-fold and 203-fold sensitivity enhancement over dye-only molecular beacons, respectively. The QDTF sensor offers a simple, robust, and truly dual approach for the precise quantification of homologous miRNAs in undiluted aCSF, holding immense potential for the early clinical diagnosis of AD.