Aptamers are an emerging class of nucleic acid molecules with applications in several fields. They are RNA or single stranded DNA molecules selected for their folding in complex tridimensional structures: their conformation, encoded in their nucleotide sequence, determines the specific interaction with diverse target molecules in solution. Aptamers bind to specific protein with high specificity and affinity, making them attractive alternatives to the commonly used monoclonal antibodies both for therapeutic and diagnostic applications. Besides lower production costs, added advantages over antibodies are their relative ease of isolation and modifications, tailored binding affinity and reversible denaturation, making them suitable candidates for use in detection systems.
We have developed a Sandwich Aptamer Microarray (SAM) for human thrombin detection immobilizing one aptamer on a glass slide (capture aptamer) and using a secondary aptamer for the detection of the analyte. This aptasensor represents a potential diagnostic system for thrombin and exploits two non-overlapping DNA antithrombin aptamers recognizing different exosites of the target protein, TBA1 and TBA2. The 15-mer aptamer TBA1 binds the fibrinogen binding site, whereas the 29-mer aptamer TBA2 binds the heparin binding domain of human thrombin. This distinct recognition pattern allows their use in tandem, since a ternary complex could possibly be formed by simultaneous recognition of thrombin.
To adapt aptamers to a defined solid phase and to a specific detection technique, appropriate chemical modifications must be introduced. In the case of the microarray, immobilization to the solid support and labeling for detection imply precise chemistries that could sensibly alter the aptamer structure. An extensive analysis on the binary complexes formation has been therefore performed in solution by Electrophoretic Mobility Shift Assay (EMSA) to verify the sandwich complex formation by a Supershift assay.
The system validated in solution was finally applied to the solid phase using an appropriate control and two different protocols for detection, resulting in an aptamer-based microarray for recognition of human thrombin with high specificity, using standard microarray slides and a fast and easy protocol. The aptamer-based biosensor could be used to analyze thrombin concentration in pathologic conditions, therefore representing a potential tool for diagnostic application
