Structure-switching aptasensor for multiplex detection of antimalarial drugs via split T7 promoter-powered transcription generation of dual-color RNA aptamers

Malaria remains a significant threat to global health, especially with the emergence of drug-resistant strains. Therefore, precise monitoring of antimalarial drug levels is crucial for assessing and managing drug resistance. Here, we report a multiplex aptasensor that enables simultaneous fluorescence detection of two antimalarial drugs (MQ and PQ) via split dsT7 promoter-mediated transcription of two-color light-up RNA aptamers. The detection mechanism relies on specific binding of target antimalarial drugs (MQ/PQ) to their aptamers, each triggering formation of intact dsT7 promoter to induce amplified transcription of its cognate RNA aptamer. The generated RNA aptamers bind their corresponding organic dyes (TO1-Biotin and MG) with high affinity, yielding 7.7-/25-fold fluorescence enhancement to enable ultrasensitive detection of MQ and PQ with limits of 0.71 ng/mL and 0.069 ng/mL, respectively. The method also exhibits high selectivity and low background signal. Furthermore, this label-free system achieves multiplexed detection of antimalarial drugs in 10-fold diluted human serum (recovery: 98.67–106.84 % for PQ and 99.89–106.19 % for MQ), demonstrating its real utility. This label-free system is adaptable to other biomarkers through aptamer replacement.