Direct Screening for Cytometric Bead Assays for Adenosine Triphosphate.

Cytometric bead assays have caught much attention because of their many exceptional advantages. Unfortunately, the immobilization of existing molecular recognition elements including monoclonal antibodies and aptamers onto solid particles may lead to the functional failure of the molecular recognition elements since they are generally obtained in free state. Herein we develop a powerful screening approach for direct and rapid discovery of aptamer based cytometric bead assays (AB-CBAs) by individually measuring the functional activity of every aptamer particles in a library and sorting them at rates of up to 108 particles per hour. The strategy is based on the transformation of molecular libraries into pools of monoclonal aptamer particles so that one individual particle displays ∼105 copies of an identical aptamer sequence. Our library design incorporates a two-color fluorescent reporter system in which changes in aptamer structure generate an optical readout, such that we can use fluorescence-activated cell sorting to rapidly and selectively separate the individual aptamer particles that exhibit large fluorescent signal change upon target binding. For demonstration, we isolated AB-CBA aptamer particles with high signaling performance for ATP after just 3 rounds of screening. We believe that the rapid and direct screening features of this strategy make it an excellent platform for generating AB-CBAs for for a wide range of important analytes.