Enzyme- and label-free cascade isothermal amplification aptasensor for the ultrasensitive detection of ochratoxin A.

Ultrasensitive detection is crucial for the early warning and intervention of risk factors, ultimately benefiting the environment and human health. Low levels of ochratoxin A (OTA) present a hidden yet significant threat, and rapid detection via high-performing biosensors is therefore essential. A cascade isothermal amplification aptasensor (CIA-aptasensor) was designed for OTA detection. On the surface of a magnetic bead probe, the OTA level was converted into positively correlated trigger cDNA through its competitive binding with OTA-Apt. The released trigger cDNA activated catalytic hairpin assembly followed by coupling with a hybridization chain reaction to achieve CIA. After adding graphene oxide and SYBR Green I, the background interference was eliminated to specifically obtain OTA-related fluorescence. The ultrasensitive limit of detection was 0.22 pg mL−1, an improvement of 1368-fold over conventional enzyme-linked aptamer sorbent assay by the same OTA-Apt, demonstrating satisfactory reliability and practicability. Thus, the CIA-aptasensor provides an enzyme- and label-free simplified homogeneous system with minimal background interference using isothermal conditions. This study provides a polymerase chain reaction–like approach for enhancing the sensitivity and performance of a biosensor, which could be extended for the application of CIA and label-free signaling strategy to other risk factors. [Display omitted] • CIA-aptasensor owned an enzyme-free, label-free, simplified and homogeneous system. • CHA and HCR-based cascade isothermal amplification was performed. • Ultrasensitive LOD was 0.22 pg mL−1, improved 1368-fold than ELASA. • Minimized background interference by GO, reflected bright fluorescence by SGI. • New guidance of performance improvements for other biosensors might recommend. [ABSTRACT FROM AUTHOR]