Step 4: Fluorescence-activated bead sorting. The flow cytometer is able to measuring fluorescence on a particle-by-particle basis. This provides an analytical readout which can be used to refine appropriate gates to determine at what level of signal the particle will be sorted into a specific containerthis is known as fluorescence-activated bead sorted (FABS). FABS has previously been used in combinatorial chemistry, including for aptamer discovery, but despite offering automation, real-time analytical output, and controllable multichannel gating, its adoption has lagged behind. We adapted FABS for selection of aptamer modifications by incubating MinE07Library with recombinant EGFR protein (extracellular domain only, amino acids 1-620) displaying an Fc-tag, and equimolar Protein A conjugated to a FITC fluorophore. Protein A-FITC binds to the Fc-tag to provide fluorescence to any bead-based aptamer to which EGFR binds. Beads with the highest affinity for EGFR would therefore be the most fluorescent. The non-labelled TG beads were first run on the FACS machine to optimise the conditions and gating (Fig. ). The aptamer library was then incubated with enough EGFR-Fc and Protein A-FITC to completely cover just 96 beads -i.e., a high stringency regime in which only the beads with the highest affinity aptamers for EGFR would acquire fluorescence. The mixture was then subjected to the first round of FABS, which selected 23,176 beads out of a total of 5,648,248 (Fig. ). The second round of FABS resulted in 4,627 sorted beads, which then underwent a third round, to give 339 beads. A fourth round, the top 170 beads were then sorted individually in well plates containing pure water (Fig. ). The aptamers were then photocleaved from the TGbeads in their wells. μ Fragments not found, or unable to assign with certainty due to short length (<5 bases), are shaded out. Their presence in the product is confirmed by the primary ion mass spectrum.