Optimization of aptamer-triggered hybridization chain reaction for rapid visual ATP detection using gold nanoparticles

Abstract

The development of fast, simple, and visual methods for detecting adenosine triphosphate (ATP) is crucial for point-of-care diagnostics and environmental monitoring. Colorimetric assays based on the integration of DNA aptamer-triggered hybridization chain reaction (HCR) with gold nanoparticles (AuNPs) offer high potential, but existing methods often suffer from prolonged detection times (e.g., >130 min). To address this limitation, this study systematically investigated and optimized the core parameters of the aptamer-triggered HCR system and its subsequent mixing conditions with AuNPs to achieve a rapid visual detection platform. The HCR kinetics were accelerated by the high concentrations of single-stranded DNA (ssDNA) components, particularly the H0 initiator, as well as by the critical presence of divalent magnesium ions (Mg²⁺), which also support the functionality of the aptamer. This optimization allowed for a degree of HCR progression comparable to the traditional 24-hour incubation to be achieved within just 60 minutes. Subsequently, recognizing the inherent trade-off between the high ssDNA concentration required for fast HCR and the AuNP dispersion stability, we optimized the mixing ratio between the HCR product and the AuNP solution. Under these optimized conditions, the assay demonstrated the capability for rapid visual detection of ATP at the 100 µM level within a total assay time of 15 minutes, representing a significant acceleration compared to previously reported methods. Further improvements in sensitivity are anticipated through future fine-tuning of AuNP parameters and the use of post-reaction salt aggregation enhancers.