Clear flat bottom black polystyrene 96 well plates

Solution properties of the anionic PBAEs were evaluated as a function of pH and time by measuring absorbance at 550 nm on a Biotek Synergy 4 plate reader in different buffers (1 mg/mL polymer in 50 mM pH 4, 6, 7, 8, or 10 buffer) in a 96 well plate (Costar clear flat bottom black polystyrene 96 well plates). Solutions (200 μL) were added to each of 3 wells. Absorbance was then measured at 550 nm at specified time points (1 h apart) and reported as an average of the triplicate measurements.

Unreacted thiol remaining after SEC purification was quantified with an Ellman’s reagent assay according to the following procedure, adapted from the manufacturer’s instructions. A sample of lyophilized, purified, polymer was dissolved in 100 mM pH 8 potassium phosphate buffer at 1 mg/mL. This solution (180 μL) was added to each of 6 wells of a 96 well plate (Costar clear flat bottom black polystyrene 96 well plates). Ellman’s reagent solution (20 μL, 4 mg/mL in 100 mM pH 8 potassium phosphate buffer) was added to each of 3 of these wells while buffer (20 μL, 100 mM pH 8) was added to each of the remaining 3 wells as a blank. Absorbance at 405 nm was read using a Biotek Synergy 4 plate reader. The triplicate measurements were averaged and the difference in absorbance between the Ellman’s reagent-containing and buffer samples compared to a calibration curve of known concentrations of MPS with Ellman’s reagent (Figure S5) to calculate the mass fraction of thiol remaining in the purified polymer samples. Samples containing less than 4 wt% thiol were used for further experiments.