Self-Assembly of Multivalent Aptamer-Tethered DNA Monolayers Dedicated to a Fluorescence Polarization-Responsive Circular Isothermal Strand Displacement Amplification for Salmonella Assay

Pathogenic bacteria are pathogens widely spread that are capable of causing mild to life-threatening diseases in human beings or other organisms. Rationally organizing the simple helical motif of double-stranded DNA (dsDNA) tiles into designed ensemble structures with architecturally defined collective properties could lead to promising biosensing applications for pathogen detection. In this work, we facilely engineered multivalent hairpin aptamer probe-tethered DNA monolayers (MHAP-DNA monolayers) and applied them to build a fluorescence polarization-responsive circular isothermal strand displacement amplification (FP-CSDA) for Salmonella assay. In this system, the MHAP-DNA monolayers were constructed based on a dsDNA tile-directed self-assembly. A FAM-labeled reporting probe (RP^FAM) with an inherent low FP signal serves as the signaling unit. The presence of target Salmonella leads to the trapping of F RP^FAM into the super DNA monolayers via a target-triggered CSDA to peel off the tethered hairpin-structured aptamer probes (HAPs) responsible for the binding of RP^FAM. As a result, the FP signal of the FAM fluorophore can be remarkably amplified due to the recycling of target Salmonella and the capacity of structural DNA materials to strongly restrict the free rotation of the FAM fluorophore but without a fluorescence quenching effect. Experimental results demonstrate that the FP assay is able to detect Salmonella with a low limit of detection (LOD) of 7.2 × 10⁰ CFU/mL and high specificity. As a proof-of-concept study, we envision our study using DNA nanoarchitecture as the foundation to modulate CSDA-based FP assays, promising to open up a new avenue for disease diagnosis, food safety detection, and biochemical studies.