Design of Label-Free DNA Light-Up Aptaswitches for Multiplexed Biosensing

We present a straightforward design approach to develop DNA-based light-up aptasensors. We performed the first systematic comparison of DNA fluorescent light-up aptamers (FLAPs), revealing key differences in affinity and specificity for their target dyes. Based on our analysis, two light-up aptamers emerged with remarkable specificity, fluorescence enhancement, and functionality in diverse environments. We then established generalizable design rules to couple the DNA FLAPs to small molecule-binding aptamers, creating 13 novel aptaswitches with reliable turn-on or turn-off aptaswitching in a dose–response manner. We developed new aptaswitches for ochratoxin A and ATP biosensing with up to a seven-fold response and low background. Finally, we demonstrated the orthogonal activity of our aptaswitch platforms. As a result, we introduce fluorescent light-up aptaswitches for one-pot detection of different targets in diverse sample matrices.