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.