Quadruple cascade strand displacement amplification reaction mediated polyfluorene derivative-based multiple FRET for synchronous determination of multiple biomarkers

Currently reported detections for biomarkers of Alzheimer’s disease (AD) were usually single-target detections, which were prone to generate misdiagnosis. This work developed quadruple cascade strand displacement amplification (QC-SDA)-mediated polyfluorene derivative-based multiple fluorescence resonance energy transfer (FRET) for simultaneous determination of AD biomarkers miRNA-16 and amyloid-beta oligomer species (AβOs). Carboxyl-functionalized poly [9,9-bis(3′-(N,N-dimethylamino)propyl)− 2,7-fluorene]-alt-2,7-(9,9-dioctylfluorene)] polymer nanoparticles ( c -PFN PNPs) served as fluorescent donor and two fluorescent dyes served as fluorescent acceptors. When the targets combined with templates to continuously trigger QC-SDA, multiple infinite cycles occurred to generate numerous triggers. The resulting triggers initiated rolling circle amplification (RCA) to generate repeated sequences, which further hybridized with helper probes and reporter probes to achieve substantial immobilization of fluorescent acceptors on c -PFN PNPs, thereby realizing multiple FRET from c -PFN PNPs to acceptors with only single excitation. As FAM and Texas Red corresponded to miRNA-16 and AβOs, the high-throughput detections of biomarkers were achieved with limit of detection of 110 aM and 1.3 fM, respectively. The c -PFN PNPs coupling FAM and Texas Red provided an ideal multiplex FRET platform for synchronous detection of multi-target. What's more, the creative incorporation of amplification strategies QC-SDA and RCA endowed multiplex FRET with high sensitivity and ease of operation for bioanalysis.