Target-Activated and Movable Toehold-Assisted Proximal Catalytic Hairpin Assembly Amplification for Low-Leakage and Ultrasensitive Fluorescent Aptamer Sensing of Glycated Hemoglobin A1c

Diabetes is a highly prevalent chronic disease worldwide, and its early diagnosis is crucial for long-term monitoring and effective management. Sensitive identification and detection of glycated hemoglobin A1c (HbA1c), a key biomarker that reliably reflects average blood glucose levels over the past 2-3 months, play an important role in the long-term monitoring of diabetes. Here, we present a fluorescent and aptamer-based highly sensitive biosensor method for HbA1c detection employing a newly designed, target-triggered, and movable toehold-assisted proximal catalytic hairpin assembly (mt-pCHA) amplification strategy. The recognition of HbA1c by the aptamer triggers the release of DNA strands to activate the mt-pCHA process, in which the assembly hairpins are arranged in close proximity on the preassembled Y-shaped scaffolds to enhance their local concentrations for accelerating reaction kinetics. Furthermore, the introduction of a movable toehold optimizes the strand displacement pathway and suppresses the background leakage while preserving the catalytic efficiency. With the HbA1c-triggered initiation of mt-pCHA, numerous quenched fluorescent hairpins are unfolded in the assembly process, leading to significantly magnified fluorescence recovery for ultrasensitive detection of HbA1c. Experimental validation demonstrates a strong linear response over the range of 5 ng/mL-50 μg/mL for HbA1c with a detection limit of 4.66 ng/mL. The assay exhibits negligible cross-reactivity toward hemoglobin (Hb) and performs reliably in diluted human whole blood hemolysate samples. These results highlight its potential as a versatile biosensing platform for the sensitive detection of low-abundance protein biomarkers.