Aptamer enzymatic cleavage protection assay for the gold nanoparticle-based colorimetric sensing of small molecules

[Display omitted] ► Homogeneous aptasensor designed for colorimetric detection of small molecules. ► Format relied on target-induced protection of aptamer from enzymatic digestion. ► Transduction attained through adsorption of DNA onto gold nanoparticles. ► Sensing platform successfully applied to tyrosinamide and adenosine detection. A label-free, homogeneous aptamer-based sensor strategy was designed for the facile colorimetric detection of small target molecules. The format relied on the target-induced protection of DNA aptamer from the enzymatic digestion and its transduction into a detectable signal through the length-dependent adsorption of single-stranded DNA onto unmodified gold nanoparticles (AuNPs). The proof-of-principle of the approach was established by employing the anti-tyrosinamide aptamer as a model functional nucleic acid. In the absence of target, the aptamer was cleaved by the phosphodiesterase I enzymatic probe, leading to the release of mononucleotides and short DNA fragments. These governed effective electrostatic stabilization of AuNPs so that the nanoparticles remained dispersed and red-colored upon salt addition. Upon tyrosinamide binding, the enzymatic cleavage was impeded, resulting in the protection of the aptamer structure. As this long DNA molecule was unable to electrostatically stabilize AuNPs, the resulting colloidal solution turned blue after salt addition due to the formation of nanoparticle aggregates. The quantitative determination of the target can be achieved by monitoring the ratio of absorbance at 650 and 520 nm of the gold colloidal solution. A limit of detection of ∼5 μM and a linear range up to 100 μM were obtained. The sensing platform was further applied, through the same experimental protocol, to the adenosine detection by using its DNA aptamer as recognition tool. This strategy could extend the potentialities, in terms of both simplicity and general applicability, of the aptamer-based sensing approaches.