Regulating the on-surface LNA probe density for the highest target recognition efficiency.

The recent emergence of on-surface LNA-based assays as potentially better alternatives over DNA-based approaches, due to enhanced sensitivity and target specificity, raises the need for the precise identification of the factors that control the performance of these assays. In this work, we investigated whether the probe density of fully modified ssLNA probes on the gold(111) surface could influence the target recognition capacity of the LNA sensing layer and illustrated simple means to control it, primarily by adjusting the salt concentration, nature of the cation, and pH of the immobilization buffer. It was observed that monovalent Na(+) could more effectively control the sensor probe density compared to bivalent Mg(2+), leading to better target recognition. Interestingly, unlike in the case of ssDNA sensor probes, the target recognition efficiency of the LNA layer at the optimum probe density was found to be almost spacer-independent, probably due to the rigidity of the LNA backbone. The optimized LNA sensor layer could discriminate single base mismatches, detect a minimum target DNA concentration of 5 nM, and sense a significant level of hybridization within a time scale of a few minutes. To our knowledge, for the first time, we identify the factors that control the on-surface LNA probe density for maximizing the performance of the LNA sensing layer.