Enhancing G-quadruplex formation in aptamers through high-annealing-temperature PCR for ultrasensitive allosteric detection of Pb2+

Developed a high-annealing-temperature PCR-assisted GO-SELEX screening strategy. • Enriched GC-rich sequences with strong G-quadruplex folding potential. • Identified a Pb 2+ -specific G4 aptamer Pb-GC-1 with high affinity and selectivity. • Constructed a Pb 2+ -responsive G4 allosteric colorimetric biosensor. • Achieved 10 nM detection limit and high recovery in real food sample testing. Heavy metal pollution, particularly from lead, poses a severe threat to global food safety. We present a novel G-quadruplex (G4) aptamers screening strategy based on high-annealing-temperature PCR-assisted graphene oxide-SELEX (HAT-GO-SELEX) for efficient development of highly specific and sensitive probes targeting Pb 2+ . By systematically elevating the PCR annealing temperature during selection, we enriched sequences with high GC content and structural stability. Analysis of the top 100 candidate sequences from high-throughput sequencing revealed significant enrichment of G-tracts, with 20% consensus G4 formation potential across multiple prediction algorithms. A Pb 2+ -specific G4 aptamer, Pb-GC-1, exhibited high affinity ( K d  = 167 nM) and strong specificity. A colorimetric biosensor based on Pb 2+ -induced G4 allosteric switching enabled sensitive detection in real food samples, achieving a detection limit of 10 nM and recoveries of 96.7–102.0%. This work presents an efficient strategy for screening functional G4-based aptamers, advancing the development of rapid detection platforms for heavy metal contaminants. Download: Download high-res image (136KB) Download: Download full-size image