Ratiometric Fluorescence Biosensing of Tandem Biemissive Ag Clusters Boosted by Confined Catalytic DNA Assembly.

The reaction kinetics and yield of traditional DNA assembly with a low local concentration in homogeneous solution remain challenging. Exploring confined catalytic DNA assembly (CCDA) is intriguing to boost the reaction rate and efficacy for creating rapid and sensitive biosensing platforms. A rolling circle amplification (RCA) product containing multiple tandem repeats is a natural scaffold capable of guiding the periodic assembly of customized functional probes at precise sites. Here, we present a RCA-confined CCDA strategy to speed up amplifiable conversion for ratiometric fluorescent sensing of a sequence-specific inducer (*) by using string green-/red-Ag clusters (AgCs and AgCs) as two counterbalance emitters. Upon recognition of *, CCDA events are operated by two toehold-mediated strand displacements and localized in repetitive units, thereby releasing * for recycled signal amplification in the as-grown RCA concatemer. The local concentration of reactive species is increased to facilitate rapider dsDNA complex assembly and more efficient input-output conversion, on which the clustering template sequences of AgCs and AgCs are blocked and opened, enabling AgCs synthesis but opposite to AgCs. Thus, the fluorescence emission of AgCs goes up, while AgCs go down. With the resultant ratio featuring inherent built-in correction, rapid, sensitive, and accurate quantification of * at the picomolar level is achieved. Benefiting from efficient RCA confinement to enhance reaction kinetics and conversion yield, this CCDA-based strategy provides a new paradigm for developing simple and diverse biosensing methodologies.