Photochrome aptamer switch assay
Date of Issue2017
School of Materials Science and Engineering
Aptamers are known to undergo adaptive conformational change upon ligand binding, causing micro-environment rearrangements that may be linked with stilbene transducers. Stilbene displays fluorescence decay upon on continuous excitation, which is sensitive to micro-environment sterics. This thesis hypothesizes that fluorescence decay based biosensing can be achieved by incorporating the molecular recognition properties of aptamer with time-dependent stilbene trans-cis photodecay. Two designs were proposed and tested according to the hypothesis: 1) “competitive binding of stilbene-analyte conjugate with analyte” and 2) “stilbene-aptamer conjugate fluorescence decay correlates to analyte binding”. Malachite green aptamer (MGA) and its ligand malachite green (MG) were chosen as a model study to test the hypothesis. Prior to hypothesis investigation, experimental conditions were established and optimized. The MGA/MG binding was found to have hydrolysis-dependent photodecay, but organic additives were found to reverse the hydrolysis and ultimately stabilize the binding. For the design 1), MG-stilbene conjugates obtained for the hypothesis testing had micromolar binding affinities with appreciable photodecay. Based on the MG-stilbene conjugates analyzed, electron donating needs to be optimized towards retaining the stilbene fluorescence decay property in MG-stilbene conjugate. In the design 2), stilbene was grafted on aptamer to fabricate the stilbene aptasensor through amine-isothiocyanate click chemistry. This conjugate maintained properties of aptamer binding and stilbene fluorescence decay, where presence of MG inhibited the fluorescence decay of MGA-SITS. The fluorescence decay of this aptasensor was also sensitive to ligand tetramethylrosamine (TMR) but presented no response to another non-binding triphenyl molecule rhodamine B (RhB), displaying good selectivity. The sensitivity of the MGA-SITS aptasensor was low for MG, when compared to TMR. Future work aims to improve the performance of the fluorescence decay based aptasensor.