Acryloxyethyl thiocarbamoyl rhodamine b

The SF concentration was adjusted
to 0.3% or to 0.03% w/v in 10 mM acetate Tris, pH 3.5, or in 5 mM
PB, pH 7.0, buffer, in the presence of the print molecule (3 nmol
of human serum albumin, Sigma-Aldrich). The final volume was 4 mL.
LAP was added at the final concentration of 0.2% or 0.04% v/w and
polymerized as reported above. Rhodamine-MIP-alb SF-NPs were prepared
in the same manner, but with the addition of 40 μL of acryloxyethyl
thiocarbamoyl rhodamine B (Sigma-Aldrich) dissolved at 0.02% w/v in
DMSO. At the end of the cross-linking process, the print molecule
was removed by the addition of Trizma free base to the NPs suspension
to reach a pH of 9.7 for 1 h, and then the MIP SF-NPs were dialyzed
with Milli-Q water 4 × 3 L under stirring. Alternatively, for
removal of the protein template, the enzyme trypsin (80 μg)
was added to the NPs for 1 h at room temperature and at the pH of
8.0, followed by acidification of the solution and dialysis. Protein
removal was controlled by SDS-PAGE electrophoresis.

NIPAm, MBAA, and potassium dihydrogen phosphate were purchased from Nacalai Tesque (Kyoto, Japan). TBAM, PVA with different degrees of polymerization and saponification, disodium hydrogenphosphate, V-50, and VA-044 were purchased from Wako Pure Chemical Co., Ltd. (Osaka, Japan). MPC, acryloxyethyl thiocarbamoyl rhodamine B, and CTAB were purchased from Sigma–Aldrich (St. Louis, MO, USA). Deionized (DI) water was obtained using a Millipore Milli-Q purification system. FAm was prepared using a previously reported procedure [9 (link)].

Bromoethanol, p-nitrophenyl
chloroformate, 3-toluenesulfonyl chloride, triethylamine, 2,4-quinolinediol,
phenylphosphonyl dichloride, p-xylylendiamine, valeroyl
chloride, calcium oxide, di-tert-butyl decarbonate,
deuterated solvents, ethyl acetate, dimethyl sulfoxide (DMSO), sodium
hydroxide (NaOH), hydrochloric acid (HCl), 2,4-dimethoxybenzyl amine,
trifluoroacetic acid (TFA), platinum on carbon (10% Pt/C), benzylamine,
benzyl alcohol, dibenzylamine, 4-dimethylaminopyridine (DMAP), 2,2′-azobis(2-methylpropionitrile)
(AIBN), n-butylamine, N,N-dimethylacrylamide (DMA), sodium bicarbonate (NaHCO₃), cholesterol, acryloxyethyl thiocarbamoyl rhodamine B, 2-mercaptoethanol,
2,2′-dipyridyl disulfide, and N,N′-dicyclohexylcarbodiimide (DCC) were obtained from Sigma-Aldrich.
Potassium sulfite, sulfur, tris(2-carboxyethyl)phosphin (TCEP), and
sodium sulfate were purchased from Acros Organics, Alfa Aesar, TCI
Chemicals, and Carl Roth GmbH+ Co. KG, respectively. N,N-Dimethylformamide (DMF), chloroform, dichloromethane
(DCM), tetrahydrofuran (THF), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate
(SMCC), and Gibco Dulbecco’s phosphate-buffered saline (DPBS)
were obtained from Thermo Fisher Scientific Inc., and methanol (MeOH),
acetone, and ethanol were obtained from Honeywell International.