The advent of personalized medicine will require biosensors capable of reliably detecting small levels of disease biomarkers. In microarrays and sensors for nucleic acids, hybridization events between surface-tethered DNA probes and the nucleic acids of interest (targets) are transduced into a detectable signal. However, target-binding ultimately occurs as a result of molecular motions and interactions between the probe and target at the nanometer scale, and common characterization methods either lack the resolution to characterize the sensors at this scale or provide only limited information about their interactions with their nanoscale chemical environment.

In this dissertation I argue that an impediment to the development of more reliable and practical biosensors is the lack of knowledge and control of the nanometer length-scale structure of biosensor surfaces, which has a profound impact on molecular recognition and reactions for detection. After reviewing the fundamental surface chemistry and structural motifs of biosensors in Chapter 1, in Chapter 2 I use electrochemical atomic force microscopy (EC-AFM) to characterize in situ a common class of model nucleic acid sensors-- thiolated DNA attached to a gold electrode which has been passivated by an alkanethiol self-assembled monolayer-- with single-molecule resolution. This level of detail allows me to observe both the conformations of individual probes and their spatial distribution at the nanoscale, then determine how these are affected by assembly conditions, probe structure, and interactions with co-adsorbates. I also determine how these nanoscale details affect the dynamic response of probes to electric fields, which have been commonly used in sensing schemes, and ultimately the ability of the surface-tethered probes to bind with target nucleic acids.

In Chapter 3, I demonstrate and optimize the nanoscale patterning of individual DNA molecules into isolated, chemically well-defined niches on the surface, and the use of these patterned probes as a single-molecule `nano-array' able to bind with target nucleic acids. Additionally, an outstanding issue is the expense of the high-quality substrates used in these studies. In Chapter 4, I discuss the development of single-crystal gold micro-plates with controllable surface chemistries as high-quality substrates for biotechnological platforms at a fraction of the cost.