Even using the best protocols for monolayer self-assembly, defects are unavoidable and found at mismatched gold crystallite domain boundaries and at large gold step edges. Surprisingly, nanoscale roughened gold provides greater monolayer stability, as smaller gold step edges force smallersized monolayer defects compared to highly smooth gold or even monocrystalline gold. However, even with the surface of a smooth gold electrode appropriately roughened to minimize monolayer defect size (Figure ), thermal desorption of the monolayer still remains a significant challenge. As further shown in Figure , moving to body temperature and/or complex biofluids further increases desorption through increased thermal energy and/or presence of foulants that provide an 'energetic bridge' to reduce the single-step energy needed for desorption (Figure ). As shown in Figure and demonstrated later in the experimental section, even small increases in van der Waals interaction between adjacent monolayer molecules, achieved by increasing alkylthiolate chain length, can greatly decrease monolayer desorption by increasing the energy step required for thermal desorption and/or desorption mediated by monolayer solubilization via interaction with proteins and other amphipathic molecules present in biofluids (Figure ). Next, the effects of electrochemical scanning on the monolayer (Figure ) are reviewed, which can both diminish and, as we show here for the first time for electrochemical aptamer sensors, also enhance monolayer stability. Firstly, there have been multiple recent demonstrations that show more gradual or reduced potential window scanning can reduce electrical stress on the monolayer. Particularly, when operating at negative potentials, the applied electric field can strain the negatively charged phosphate backbone of the aptamer away from the electrode surface. We additionally show here in the experimental results that, while operating at negative potentials can accelerate monolayer desorption, that same cathodic scanning also mitigates the oxidation of alkylthiolate molecules, setting the stage for optimized scanning as shown in Figure . While literature has demonstrated that attaching single molecules to gold with multiple thiol groups reduces their desorption due to thermal or electric-field effects, we will not test those methods herein because in most examples poorer packing-density of the resulting monolayer would increase electrochemical background current due to higher capacitance and increased oxygen reduction at the surface.