Design of Label-Free\nDNA Light-Up Aptaswitches for\nMultiplexed Biosensing

We present a straightforward design\napproach to develop\nDNA-based\nlight-up aptasensors. We performed the first systematic comparison\nof DNA fluorescent light-up aptamers (FLAPs), revealing key differences\nin affinity and specificity for their target dyes. Based on our analysis,\ntwo light-up aptamers emerged with remarkable specificity, fluorescence\nenhancement, and functionality in diverse environments. We then established\ngeneralizable design rules to couple the DNA FLAPs to small molecule-binding\naptamers, creating 13 novel aptaswitches with reliable turn-on or\nturn-off aptaswitching in a dose–response manner. We developed\nnew aptaswitches for ochratoxin A and ATP biosensing with up to a\nseven-fold response and low background. Finally, we demonstrated the\northogonal activity of our aptaswitch platforms. As a result, we introduce\nfluorescent light-up aptaswitches for one-pot detection of different\ntargets in diverse sample matrices.