Displacement Enzyme Linked Aptamer Assay

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Abstract
Immense effort has been placed on the realization of immunoassays exploiting displacement of a suboptimum target, due to the ease of use and applicability to immunochromatographic strips and immunosensors. Most of the efforts reported to date focus on the use of a suboptimal target that is displaceable by the target toward which the antibody has higher affinity. Limited success has been achieved due to difficulty in obtaining suboptimal targets to which the antibody has enough affinity to bind while at the same time having lower levels of affinity in comparison to the target to facilitate displacement. Aptamers are synthetic oligonucleotides specifically selected to bind a certain target. Thanks to their high affinity and sensitivity, aptamers appear as alternative candidates to antibodies for analytical devices and several enzyme-linked aptamer assays and aptasensors have been reported. Aptamers, in contrast to antibodies, require the formation of a three-dimensional structure for target binding and can thus be anticipated to have a much higher affinity for binding its target rather than a modified form of the target (e.g., enzyme-labeled target). This phenomenon can be exploited for the development of a displacement assay, using enzyme-labeled target as a suboptimal displaceable molecule. Here, we report the first demonstration of the exploitation of an aptamer in an extremely rapid and highly sensitive displacement assay. Surface plasmon resonance studies demonstrated the thrombin-binding aptamer to have a lower affinity for enzyme-labeled thrombin than unmodified thrombin, with respective K(D) of 1.1 x 10(-8) and 2.9 x 10(-9) M. The assay is extremely rapid, requiring only 10 min for completion, and exhibits a detection limit lower than that obtainable with competitive enzyme-linked aptamer assays and comparable to that of hybrid aptamer-antibody assays. Optimal storage conditions for precoated microtiter plates (consisting of coated aptamer and captured labeled target) were elucidated, and the results demonstrated their amenability to long-term storage, facilitating commercially viable displacement enzyme-linked aptamer assays that simply require sample addition, with a total assay time, including color development, of 30 min.