Aptamer sandwich assays: label-free and fluorescence investigations of heterogeneous binding events
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We studied aptamer binding events in a heterogeneous format using label-free and fluorescence measurements for the purpose of developing an aptamer-based sandwich assay on a standard microtiter plate platform. The approach allowed visualization of the underlying aptamer immobilization and target binding events rather than relying on only an endpoint determination for method optimization. This allowed for a better understanding of these multi-step assays and optimal conditions specific to aptamers. α-thrombin was chosen as a prototypical analyte as two well-studied aptamers (15 and 29-mer) binding distinct epitopes are available. The Corning Epic® system, which utilizes a resonance waveguide diffraction grating in a 384-well microtiter plate format, was employed to measure relative immobilization and binding levels for various modified aptamers. Parameters investigated included the effects of aptamer orientation, label orientation, spacer length, spacer type, immobilization concentration, and binding buffer. Most notably, the 15-mer aptamer was preferable for capture over the 29-mer aptamer and aptamers with increasing poly(dT) spacer length between the biotin modification and the aptamer yielded decreased immobilization levels. This decreased immobilization resulted in increased α-thrombin binding ability for 15-mer aptamers with the poly(dT) spacer. Fluorescence measurements of fluorescein-labeled 29-mer aptamers with varying spacers were used to visualize sandwich complex formation. Using both label-free and traditional fluorescence measurements, an in-depth understanding of the overall assay was obtained, thus the inclusion of label-free measurements is recommended for future method development.
KeywordsLabel-free Aptamer Fluorescence Epic® Sandwich assay Immobilization
limit of detection
Tris–ethylene diamine tetraacetic acid
The authors are grateful to Corning Incorporated for their installation of the Epic® system at Cornell University and discussions with Alice Gao, Ph.D. and Ravi Marala, Ph.D. We also thank Cynthia Kinsland, Ph.D. and Jeffery Mattison of the Protein Production and Characterization Facility at Cornell University for their training and technical assistance on the Epic® system. This project was funded in part by the CD4 Initiative, Imperial College, London.
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