Nanoscale spatial distribution of thiolated DNA on model nucleic acid sensor surfaces

The nanoscale arrangement of the DNA probe molecules on sensor surfaces has a profound impact on molecular recognition and signaling reactions on DNA biosensors and microarrays. Using electrochemical atomic force microscopy, we have directly determined the nanoscale spatial distribution of thiolated DNA that are attached to gold via different methods. We discovered significant heterogeneity in the probe density and limited stability for DNA monolayers prepared by the backfilling method, that is, first exposing the surface to thiolated DNA then "backfilling" with a passivating alkanethiol. On the other hand, the monolayers prepared by "inserting" thiolated DNA into a preformed alkanethiol monolayer lead to a more uniformly distributed layer of DNA. With high-resolution images of single DNA molecules on the surface, we have introduced spatial statistics to characterize the nanoscale arrangement of DNA probes. The randomness of the spatial distribution has been characterized. By determining the local densities surrounding individual molecules, we observed subpopulations of probes with dramatically different levels of "probe crowding". We anticipate that the novel application of spatial statistics to DNA monolayers can enable a framework to understand heterogeneity in probe spatial distributions, interprobe interactions, and ultimately probe activity on sensor surfaces.