Label-Free Fluorescent Aptasensor for Small Targets via Displacement of Groove Bound Curcumin Molecules.

Signal transduction based on fluorescence is one of the most common optical aptasensors for small molecules. Sensors with a number of unique features including high sensitivity, low cost, and simple operation can be constructed easily. However, the label-free fluorescent approach is limited to synthetic dyes that bind strongly to the aptamer sequence and result in a diminished sensor operation with high detection limits. In this study, we report the use of curcumin as a fluorescent probe to signal aptamer/small target binding events. A substantial enhancement in curcumin's fluorescent emission was observed when bound into the grooves of vitamin D3 (VTD3) binding aptamer, as an example. However, the introduction of the target molecule causes the aptamer to undergo a conformational change that favors complexing the target molecule over binding the curcumin dye. The sensor was able to detect VTD3 down to 1 fM concentration in buffer solutions and extracted blood samples, operate at a wide dynamic range, and discriminate against potential biological interfering molecules including VTD2. The operation of the curcumin based fluorescent sensor is at least six orders of magnitude more sensitive than a VTD3 sensor constructed with the synthetic dye SYBR Green I. The generality of the reported label-free approach was applied with a previously isolated 75-mer bisphenol-A (BPA) aptamer, confirming that the reported sensing strategy is not confined on a particular aptamer sequence. Our work not only reports a novel sensor format for the detection of small molecules, but also serves fluorescent sensor's most pressing need being novel fluorophores for multiplex targets detection.

Colorimetric Aptasensor of Vitamin D3: A Novel Approach to Eliminate Residual Adhesion between Aptamers and Gold Nanoparticles.

Colorimetric aptasensors based on gold nanoparticles (AuNPs) commonly feature ssDNA probes nonspecifically adsorbed to surface gold particles. A major limitation of this versatile method is the incomplete dissociation of the adsorbed nontarget binding segments of the aptamer sequence upon target binding. This results in weak or nonexistent sensor performance by preventing the particles from aggregating when the optimized salt concentration is added. Rather than removing the nonbinding nucleotides flanking the binding region of the aptamer, proposed herein is an alternative strategy, simply introducing a centrifugation and resuspension step after target recognition that eliminates residual binding between the aptamer and the surface of the particles. The performance of two different vitamin D3 (VTD3) aptamers were tested. The method enhanced the performance of the sensor that used the higher detection limit (1 µM) aptamer by fourfold. The superiority of the proposed method became apparent in a nonworking colorimetric sensor became a highly sensitive sensor with a one nanomolar detection level and excellent discrimination against potential interfering molecules including VTD2 when the centrifugation and resuspension process was implemented. The level of VTD3 in human blood was determined colorimetrically after extraction with n-hexane. The results were in agreement with those obtained by HPLC. The proposed method could be applied to aptamers targeting small molecules with no need to reprocess the SELEX-isolated sequence by knowing the binding region and removing the flanking primers.