Enhancement of aptamer microarray sensitivity through spacer optimization and avidity effect

This work aims for ultrasensitive detection of target proteins in complex biological matrixes based on aptamer microarrays. Two extensively studied aptamers (HTQ and HTDQ) that bind distinct epitopes of thrombin are chosen for the microarray study. Although HTQ and HTDQ have nanomolar to subnanomolar affinities, it is found that either aptamer when applied directly has difficulty in detecting a few nanomoles per liter thrombin in the presence of a 10- or 100-fold (w/w) excess of serum total protein (STP). By investigating dodecyl (12-carbon) and oligodeoxythymidine (oligo(dT)) spacers, we observe both spacers enhance the microarray signal response, but oligo(dT) is strikingly better than dodecyl. Moreover, we discover that a microarray spot coprinted with the two distinct aptamers (HTQ and HTDQ) functions like a bivalent molecular construct and exhibits an avidity effect. With the synergy of oligo(dT) spacers and the avidity effect, detection of picomolar-range thrombin in the presence of either 10% unlabeled serum or a 10 000-fold excess of labeled serum total protein is achieved. It corresponds to a 100-1000-fold sensitivity enhancement as compared to using an individual aptamer without a spacer.