Cell Probe Cocktail Enables Ratiometric miRNA Detection with Enhanced Sensitivity and an Extended Dynamic Range

Sensitive and wide-range detection of miRNA is crucial for monitoring early stage and progressive diseases. However, achieving both high sensitivity and a broad response range in a single sensing platform remains challenging due to the inherent trade-off between these properties. In this study, we develop a novel cell probe cocktail integrated with nicking endonuclease-assisted strand displacement amplification (SDA) for the ratiometric detection of miRNA, enabling concurrent enhancement of sensitivity and expansion of the dynamic range. The counting probes were constructed by conjugating suspended cells with aptamer-functionalized magnetic nanoparticles. A cell probe cocktail system was thus created by combining two types of these counting probes. Leveraging the programmability of DNA, target recognition-induced conformational changes enable the regulation of cell probe assembly and disassembly by modulating the binding of aptamers to cell surface receptors, thereby yielding sensitive, wide-dynamic-range ratiometric readouts. As a proof of concept, ratiometric detection of miRNA-21 over a concentration range of 0.2-100 nM was successfully achieved, with a limit of detection (LOD) of 0.038 nM. The inherent programmability of DNA underscores the versatility of this cell probe cocktail strategy, enabling adaptive sensing across diverse DNA-based architectures.