LAUSR

DNA aptamers have emerged as promising probes for challenging analytes that cannot be easily detected by conventional probes, including small-molecule targets. Among the different signal transduction approaches, gold nanoparticle (AuNP) aggregation assays have been widely used to generate a colorimetric response from aptamer-target interactions. This sensor design relies on the competition between the aptamer adsorbing to the AuNP surface versus interacting with the target, whereby target binding reduces the number of adsorbed aptamers that destabilizes AuNPs toward salt-induced aggregation, thereby inducing a color change. However, this thermodynamic framework overlooks the potential influence of interaction kinetics of different aptamer conformations with AuNP surfaces and with targets in solution or near surfaces. Here, we show that aptamers become more strongly adsorbed on AuNPs over time, and these trapped aptamers are less responsive toward the target analyte. By varying the sequence of addition in sensing assays, we demonstrate that these interaction kinetics have a significant effect on the sensor response and thereby produce an effective sensor for methamphetamine (meth) at biologically relevant levels in oral fluids. Along with underpinning new tools for assay development, this new knowledge also highlights the need for aptamer selection strategies that evolve aptamer sequences based on the functionality that they need to exhibit in an actual sensor.