Fluorescent aptasensors are reliant on static fluorescence intensity measurements, which suffer drawbacks such as background interference and laborious separation procedures. A unique aptasensor based on the photochrome aptamer switch assay (PHASA) has been developed that is independent of background fluorescence, requires no analyte separation, and allows rapid quantification within seconds. Malachite green aptamer (MGA) conjugated with a water-soluble stilbene on the MGA 3′ C38 terminus was chosen for building the proof-of-concept aptasensor. In the presence of malachite green and tetramethylrosamine ligands, the rate of the stilbene fluorescence decay was found to be linearly dependent on the ligand concentration. Molecular dynamic simulation suggests hydrogen bonding between stilbene sulfonates and neighboring nucleotides is the primary mechanism responsible for rate changes in stilbene photoisomerization. Analysis of the apparent fluorescence decay rate (kapp) versus analyte concentration gives a limit of detection (LOD) of 2 μM for MG and 0.6 μM for TMR. This aptasensor design opens up a new sensing mode, which is promising for rapid development of SELEX generated molecular recognition elements.
- Fluorescence decay
- Malachite green