Construction of multivalent aptamer-based fluorogenic DNA nanostructures for label-free fluorescent biosensors

Fluorescent light-up aptamers (FLAPs) are attractive signaling tools due to their label-free fluorescence and programmability. However, DNA-based FLAP sensors are often constrained by low fluorogenic activation efficiency, limited intrinsic target responsiveness, and insufficient signal amplification. In this study, we integrated a DNA-based FLAP (DAP) with a three-branched DNA (TBD) scaffold to construct a multivalent FLAP nanostructure (DAP-TBD) that exhibited enhanced fluorogenic activation toward Auramine O (AO). Compared with monovalent DAP, the trivalent DAP-TBD showed a 3.3-fold increase in fluorogenic activation efficiency, indicating a clear multivalent effect. The system adopts a modular architecture in which DAP functions as a replaceable FLAP model. Using a target-triggered catalytic DNA assembly (CDA) circuit composed of three rationally designed hairpin DNAs, multiple DAP-TBD nanostructures are assembled isothermally, enabling target-triggered signal generation and amplification without the need for labeled probes. The CDA-based DAP-TBD system selectively detects target single-stranded DNA with a detection limit of 235 pM. Furthermore, coupling the DAP-TBD system with an exonuclease-assisted hairpin and an aptazyme enabled label-free detection of microRNA and adenosine triphosphate, demonstrating the versatility of the system as a signal sensor that can interface with diverse upstream transducer modules. The system requires no fluorophore or quencher labeling, relies on only three unmodified DNA strands, and offers programmability, modularity, and cost-effectiveness. Overall, this study presents a multivalent DNA-based FLAP nanostructure and provides a versatile strategy for developing label-free fluorescent biosensors.