Ultrasensitive DNAzyme-Based Ca²⁺ Detection Boosted by Ethanol and a Solvent-Compatible Scaffold for Aptazyme Design

Functional DNA includes aptamers and DNAzymes, and metal ions are often important for achieving the chemical functions of such DNA. Biosensors based on functional DNA have mainly been tested in aqueous buffers. By introducing organic solvents with much lower dielectric constants, the interaction between metal ions and DNA can be significantly enhanced, and this might affect the performance of DNA-based biosensors. In this work, the effect of ethanol on the activity of the EtNa DNAzyme was studied for Ca²⁺ detection. With 30 % ethanol, the sensor has a detection limit of 1.4 μm Ca²⁺ , which is a 16-fold improvement relative to that in water. This EtNa DNAzyme is unique because other tested DNAzymes are all inhibited by 50 % ethanol. Finally, by using the EtNa DNAzyme as a scaffold, the adenosine monophosphate (AMP) aptamer was inserted to construct an aptazyme, which allowed the measurement of AMP in ethanol. In summary, this study has reported the most sensitive DNA-based sensor for Ca²⁺ , and its sensitivity and selectivity can approach those of proteins or small-molecule ligands. This work also provides a way to measure aptamer binding in organic solvents.