PEG-based Microgel Synthesis with Biotin

A microgel precursor solution containing 5% w/v PEG-4MAL (20kDa, Laysan Bio) and 1.0 mM biotin-PEG-thiol (1 kDa, Nanocs) was reacted for 15 min in PBS. This precursor was dispersed into droplets and subsequently crosslinked within mineral oil (Sigma) containing 2% SPAN80 (Sigma) and a 1:15 emulsion of 30 mg/mL dithiothreitol (Sigma) on a microfluidic chip, as described previously ^{23 (link)}. Control microgels that did not contain biotin-PEG-thiol were also synthesized. After washing microgels 5 times by centrifugation in 1% bovine serum albumin (Sigma) in PBS, 10⁴ microgels were incubated with varying concentrations of a streptavidin-AlexaFluor488 conjugate for 30 min in 500 µL PBS, and were washed 5 times by centrifugation to remove unbound SA. Microgels from each sample were placed in a 96-well plate and fluorescence was measured on a plate reader (Perkin Elmer HTS 7000). Biotin and control microgels were also synthesized with a covalently bound peptide (GRGDSPC)-AlexaFluor594 conjugate for microgel visualization, and were fluorescently imaged to confirm biotin-specific SA immobilization.