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

ACS Sens. 2025 Jan 24;10(1):246-253. doi: 10.1021/acssensors.4c02331. Epub 2024 Dec 20.

Abstract

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.

Keywords: DNA; aptamers; aptaswitch; biosensors; fluorescence; metabolites; multiplexing.

MeSH terms

  • Adenosine Triphosphate* / analysis
  • Aptamers, Nucleotide* / chemistry
  • Biosensing Techniques* / methods
  • DNA* / chemistry
  • Fluorescent Dyes / chemistry
  • Ochratoxins* / analysis

Substances

  • Aptamers, Nucleotide
  • Ochratoxins
  • ochratoxin A
  • Adenosine Triphosphate
  • DNA
  • Fluorescent Dyes