Methods

Polymers containing chain-transfer agent (CTA) and various amounts of trehalose glycopolymer and pNIPAM were synthesized using RAFT synthesis and confirmed by NMR (MWs: 8-38 kDa)

Four polymers (all with carboxyl end group CTA) were chosen to test with alkaline phosphatase (AP): pNIPAM, trehalose glycopolymer, and two copolymers: one with pNIPAM and trehalose glycopolymer in blocks and the other with pNIPAM and trehalose glycopolymer randomly intermixed

AP (25 µg/mL in microcentrifuge tube) was mixed with each of the chosen polymers (2.5 mg/mL, approx. 100x the concentration of AP) separately in microcentrifuge tubes in triplicate, flash frozen with liquid nitrogen, and stored at -80℃ [1]

A microcentrifuge tube with just AP in deionized water and a microcentrifuge tube with AP and trehalose sugar (unpolymerized) were tested as controls in triplicate

3 more sets of these samples (72 tubes total) were created for each time period (0, 24, 48, and 72hrs) to be incubated at one of various temperatures (room, 37℃, 55℃, and 70℃)

Repeat for each temperature

An incubator was used to incubate samples at 37℃, a water bath was used to incubate at 55℃, and an oven was used to incubate at 70℃

The enzymatic activity of each set was measured by colorimetric assay using Quanti-Blue (1.168 mg/mL 5-bromo-4-chloro-3-indolyl phosphate (BCIP) p-toluidine salt (VWR) in 1M aqueous diethanolamine) [4]

Each sample was pipetted (20 µL) on a microwell plate and reacted with Quanti-Blue (180 µL), then analyzed using a UV-vis spectrometer

The absorbance data was averaged and normalized to the “AP only” value at 0 hrs for each temperature

Statistically significant differences were determined using two-tailed, two sample t-tests assuming unequal variances at 95% confidence using the absorbances for “AP only” at 0 hrs as the reference for each temperature