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Abstract Solid surfaces that are immobilized with DNA molecules underlie an array of biotechnological devices. These surfaces may also mediate the self‐assembly of hierarchical DNA nanostructures. However, a number of fundamental questions concerning the structure–function relationship of these biointerfaces remain, including how these DNA probe molecules organize on the surface and how the spatial organization influences molecular recognition kinetics and interfacial affinity of these DNA molecules at the regime where crowding interactions are important (1–10 nm). This mini‐review covers recent advances in understanding this structure–function relationship by spatially resolving surface hybridization events at the single‐molecule level. Counterintuitive cooperative effects in surface hybridization are discussed and as is how modeling these cooperative effects can be used to predict the hybridization kinetics of a prototypical DNA sensor. Future opportunities in using mechanistic understanding to improve the performance and reliability of DNA sensors and form hierarchical supramolecular structures are also discussed. 

The spatial arrangement of target and probe molecules on the biosensor is a key aspect of the biointerface structure that ultimately determines the properties of interfacial molecular recognition and the performance of the biosensor. However, the spatial patterns of single molecules on practical biosensors have been unknown, making it difficult to rationally engineer biosensors. Here, we have used high-resolution atomic force microscopy to map closely spaced individual probes as well as discrete hybridization events on a functioning electrochemical DNA sensor surface. We also applied spatial statistical methods to characterize the spatial patterns at the single molecule level. We observed the emergence of heterogeneous spatiotemporal patterns of surface hybridization of hairpin probes. The clustering of target capture suggests that hybridization may be enhanced by proximity of probes and targets that are about 10 nm away. The unexpected enhancement was rationalized by the complex interplay between the nanoscale spatial organization of probe molecules, the conformational changes of the probe molecules, and target binding. Such molecular level knowledge may allow one to tailor the spatial patterns of the biosensor surfaces to improve the sensitivity and reproducibility.