DNA sequences that can bind selectively and specifically to target molecules are known as aptamers. Normally such binding analyses are performed using soluble aptamers. However, there is much to be gained by using an on-chip or microarray format, where a large number of aptameric DNA sequences can be interrogated simultaneously. To calibrate the system, known thrombin binding aptamers (TBAs) have been mutated systematically, producing large populations that allow exploration of key structural aspects of the overall binding motif. The ability to discriminate between background noise and low affinity binding aptamers can be problematic on arrays, and we use the mutated sequences to establish appropriate experimental conditions and their limitations for two commonly used fluorescence-based detection methods. Having optimized experimental conditions, high-density oligonucleotide microarrays were used to explore the entire loop-sequence-functionality relationship creating a detailed model based on over 40000 analyses, describing key features for quadruplex-forming sequences. © 2009 The Royal Society of Chemistry.