The advancement of detection modalities with extremely high resolution and low background levels has also made it possible for low-affinity binding reagents to achieve high detection sensitivity 2, 6, 7. On the other hand, low-affinity reagents with fast association and dissociation rates have been used in real-time monitoring applications 3, 4, 5, because they can resolve the dynamic changes in their target analyte level. High-affinity recognition molecules with low dissociation constants ( K d) are sought for point-of-care applications to enable rapid binding events and low limits of detection. For example, the binding affinity between a target analyte and its specific biorecognition element is the determinative factor that defines the dynamic range of a biosensor 1, 2. Moreover, molecules with well-defined binding affinities for a specific target-thus called affinity reagents-are essential for many applications, including biosensing, diagnostics, imaging and therapeutics. Precise control over the binding affinities between biomolecules is one of the critical means by which nature allows thousands of types of biomolecule to operate in tandem with molecular-level control in heavily crowded environments. Using human myeloperoxidase as a target, we demonstrate that aptamers with dissociation constants spanning a 20-fold range of affinities can be identified within one round of Pro-SELEX. Using the Pro-SELEX workflow, we were able to investigate the binding performance of individual aptamer candidates under different selective pressures in a single round of selection. Here we introduce Pro-SELEX, an approach for the rapid discovery of aptamers with precisely defined binding affinities that combines efficient particle display, high-performance microfluidic sorting and high-content bioinformatics. Generating aptamers with different binding affinities is desirable, but systematic evolution of ligands by exponential enrichment (SELEX), the standard for aptamer generation, is unable to quantitatively produce aptamers with desired binding affinities and requires multiple rounds of selection to eliminate false-positive hits. Aptamers are being applied as affinity reagents in analytical applications owing to their high stability, compact size and amenability to chemical modification.
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