Electrochemical DNA biosensors, which immobilize nucleic acid capture probes onto the electrode surfaces, hold significant potential in point-of-care diagnostics due to their abilities in highly selective, label-free, and miniaturized detection of a range of biomarkers in complex biofluids. An outstanding challenge is that the influences of the biointerface are complex and difficult to predict, hampering the effort to engineer biosensors with predictable performance. One aspect that has been particularly poorly understood is how the nanoscale lateral organization of probe and captured target molecules influences molecular recognition. Although the spatial arrangement of target and probe molecules on the biosensor is likely a major determinant of the properties of interfacial molecular recognition and the performance of the biosensor, the spatial patterns of single molecules on practical biosensors have been unknown. Here we have used high resolution, in situ 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.

Single molecule profiling of molecular recognition at a model electrochemical biosensor

2019 Spring National Meeting

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