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For improving aptamer-ligand binding we have developed a screening system that defines optimal binding buffer composition. Using multiplex assays, one buffer system is needed which guarantees the specific binding of all aptamers. We investigated nine peer-reviewed DNA aptamers. Non-specific binding of aptamers is an obstacle. To address this, we investigated 16 proteins as specificity controls bound covalently to encoded microbeads in a multiplex assay. Increasing the NaCl concentration decreased the binding for all aptamers. Changing pH values by one unit higher or lower did not influence the aptamer binding significantly. However, pH &lt; 5 led to non-specific binding for all aptamers. The  Pf LDH-aptamer selected in the absence of divalent cations exhibited doubling of its binding signal by the addition of Ca 2+  and Mg 2+ . We confirmed Ca 2+  and Mg 2+  dependency of the aptamers for streptavidin and thrombin by observing a 90% and 50% binding decrease, respectively. We also achieved a doubling of binding for the streptavidin aptamer when replacing Ca 2+  and Mg 2+  by Mn 2+ . A buffer suitable for all aptamers can have considerable variations in pH or ionic strength, but divalent cations (Ca 2+ , Mg 2+ , Mn 2+ ) are essential.

A multiparametric fluorescence assay for screening aptamer–protein interactions based on microbeads