An entropy–enthalpy co-driven independent dual-mode biosensor

The rapid and reliable quantitative detection of biological targets encounters challenges such as limited driving forces in reactions, low overall strand utilization, and the synthetic complexity of multi-modal materials. To address these issues, the design concept "entropy-enthalpy coupling with dual-mode decoupling" is introduced for the first time. Using miRNA-21 as a model analyte, an entropy-enthalpy co-driven decoupled dual-mode biosensor was developed by integrating independent photoelectrochemical (PEC) and fluorescent (FL) modules to assess its feasibility. Specifically, an output strand was designed within a three-stranded substrate/output complex, in which the terminal bases form hairpin structures through complementary pairing. This configuration enables the establishment of an entropy-enthalpy coupling driving force-increased entropy for the substrate strand and decreased enthalpy for the output strand-during cyclic amplification reactions. Additionally, by conjugating the substrate, fuel and output strands separately to magnetic beads, photosensitive materials, and fluorophores, complete decoupling of the PEC and FL modes was achieved, allowing independent PEC and FL signal outputs. In the developed biosensor, the device demonstrated well-defined linear correlation for PEC and FL within the 10 aM to 10 nM range, and the detection limits for miRNA-21 were established at 4.2 aM (PEC) and 5.3 aM (FL). Experimental results demonstrated the substantial potential of the "entropy-enthalpy coupling with dual-mode decoupling" framework for rapid and reliable quantitative detection of biological targets, while also providing new design paradigms for sensors in fields such as food safety and agriculture.