2.1 Experimental materials and method
Materials. Tert-butyl chlorodimethylsilane (TBDMSCl, 99%), α-bromoisobutyric acid (BiBA, 98%), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 98%), α-bromoisobutyryl bromide (BiBB, 98%), tris(2-aminoethyl) amine (TREN, 98%), tetrabutylammonium fluoride (TBAF, 1 M solution in THF containing ca. 5% H2O), 2,2’-bipyridine (Bpy, 99%), copper (I) bromide (purified before use), copper (II) bromide (99%), diisopropylethylamine (DIPEA, 99%), acetonitrile (MeCN, 99.9%, superdry, water ≤ 30ppm), N,N-dimethylformamide (DMF, HPLC grade) and tetrahydrofuran (THF, 99.9%, superdry, stabilized with 250ppm BHT) were provided by J&K Chemical Ltd. (Shanghai, China). Dopamine hydrochloride (DOPA·HCl, 98%), L-β-(3,4-dihydroxyphenyl)alanine (L-DOPA, 98%), N-hydroxysuccinimide (NHS, 98%), dicyclohexylcarbodiimide (DCC, 99%), phosphate buffer saline (PBS, pH 7.4, 150 mM, 138 mM NaCl, 2.7 mM KCl), and sulfobetaine methacrylate (SBMA, >98%) were purchased from Sigma-Aldrich (Shanghai, China). Imidazole (98%), dichloromethane (DCM, 99%), trimethylamine (N(Et)3, 99%), deuterated chloroform (CDCl3, 99%), deuterated methanol (CD3OD, 99%), deuterated water (D2O, 99%), concentrated sulfuric acid (98%), and hydrogen peroxide (30%) were obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Silicon wafer (10mm×10mm) was purchased from Shanghai Guang Wei Electronic Material Ltd. Peroxidase-Labeled Antibody to Human IgG (PLA-IgG) and fluorescein isothiocyanate conjugated bovine serum albumin (FITC-BSA) was obtained from MOLBASE. Phosphate buffered saline (PBS) solution (0.01 M, pH 7.4) was prepared from PBS powder purchased from Sigma-Aldrich. All water used in the experiment was obtained from ultrapure water equipment.
Synthesis of sin-DOPA-PSBMA. The preparation of sin-DOPA-PSBMA was the same as the previously reported by Jiang18, 19. Fabrication of sin-DOPA-PSBMA was in classical methanol/H2O system using the ATRP method20-23.
Synthesis of tri-DOPA-PSBMA. Different from the polymerization of sin-DOPA-PSBMA in the methanol/H2O system, the synthesis of tri-DOPA-PSBMA was stirred in DMF/H2O system as the tri-DOPA-Br initiator was undissolved in the methanol/H2O system. Briefly, tri-DOPA-Br powder (43.14 mg, 0.0238 mmol), CuBr (11.3 mg, 0.0786 mmol)/ CuBr2(2.8 mg, 0.0125 mmol) and Bpy (31.3 mg, 0.2 mmol) were added to a Schlenk flask and filled with N2 after degassed three times. Then degassed DMF (4 mL) was added to the system via a syringe and kept stirring for 10min at r.t. After that, a degassed solution of SBMA (1.397 g, 5 mmol) in H2O/DMF (3 mL / 6.5 mL) was injected into the reaction system via a syringe and placed into a 20 °C incubators for fifteen hours. Then the flask was put into liquid N2 to quench the reaction and thawed after a while. The collected polymer precipitate was dissolved in water and dialyzed in water for three days. The white powder was obtained after lyophilization. Then the obtained powder with the protection of TBDMSCl (0.1128 mmol) was reacted with TBFA (0.282 mmol) in THF for twenty hours under an N2 atmosphere. Unprotected white powder tri-DOPA-PSBMA was collected after rinsing three times with THF and dried in vacuum at room temperature.
In this work, DOPA-PSBMA with three different degrees of polymerization were prepared respectively by tuning the feed ratios of the initiator and the monomer (Table S4 ).
Fabrication of PSBMA coated substrates via “grafting to” method. The silicon wafer was sonicated with water, ethanol, and water in turn at a power of 30 Hz, then dried with N2. The dried silicon wafer was immersed in a boiling ‘piranha’ solution (concentrated sulfide acid/hydrogen peroxide = 7/3) for 1 h. (Caution: Piranha solution is extremely dangerous and easily exploded when came across with organic material, and it’s forbidden to contact with boiling solution beaker directly unless it’s cold). Then the substrates were rinsed and sonicated with water at least three times. The cleaned silicon wafers were dipped in the sin-DOPA-PSBMA or tri-DOPA-PSBMA solutions in tris-buffer (10 mM, pH 8.5) at a polymer concentration of 2 mg/mL and incubated in a thermostatic oscillator at 25 °C. The “grafting to” protocol used in the experiment was the optimal condition24, 25. Then the substrates were rinsed with PBS buffer three times and then with water three times and dried with N2.
Characterizations.1H NMR and 13C NMR spectra were collected using a Brüker AV-400 nuclear magnetic resonance spectrometer in CD3OD, CDCl3, or D2O. The ESI mass spectra were measured on a Waters orthogonal acceleration time of flight mass spectrometer. Hydrodynamic diameters of DOPA-PSBMA polymers in an aqueous solution were carried out using dynamic light scattering (DLS) (Malvern Zetasizer NanoZS, UK). The water contact angle was recorded at least three different areas on the substrate surfaces with a contact angle goniometer (Zhong Chen POWEREACH, Shanghai). The X-ray photoelectron spectroscopy (XPS) of modified substrates was performed using a Perkin-Elmer PHI 5000C ESCA system with an Al Kα micro-focusing X-ray source at a power of 250 W and detected a depth of about 10 nm.
Protein adsorption test with ELISA and FITC-BSA protein adhesion. Protein adsorption on the substrates was performed using an enzyme-linked immunosorbent assay (ELISA) on a microplate spectrophotometer (Bio TeK, USA) in a standard protocol18, 26, 27. Briefly, the samples were placed into a 24-well tissue culture plate containing 1 mL of 1μg/mL PLA-IgG mixed with PBS buffer in individual wells and incubated for one h in the thermostatic oscillator. All samples were transferred into new wells after being rinsed five times with 1mL of PBS and once with 1 mL of water. Then to each well was added 1 mL of 1 mg/mL o-Phenylenediamine (OPD) dissolved in 0.1 M citrate-phosphate buffer (pH 5.5) containing 0.03% hydrogen peroxide. 1 mL of 1 M HCl was added to each well to stop the reaction after 15 min without light. 150 μL of solution in each well was added to a 96-well plate, and the light absorbance at 492 nm was measured on a microplate spectrophotometer. The absorption value of the uncoated silicon wafer was viewed as a standard 100% to compare with the relative absorbance of the others. Each kind of acquired absorbance was performed on at least three silicon wafers.
The substrates were incubated in each well of a 24-well plate with 0.5 mL of 0.25 mg/mL FITC-BSA in PBS buffer for two h at 37 °C in darkness. After five times rinsed with 1 mL of PBS buffer and once with 1 mL of water, the substrates were dried with N2. The adhesive FITC-BSA was detected using Laser Scanning Confocal Microscopy (LSCM) (NIKON, Japan) at λexcitation = 488 nm/λemission = 520 nm.
Stability of Coated Substrates in PBS Buffer. The evaluation method based on the stability of modified silicon wafers was used, as previously reported by Minko28. Briefly, the coated substrates were placed in a 24-well plate with 2 mL PBS buffer in each well at r.t. The substrates were taken out after being incubated at different times and immersed in 0.5 mL of 0.25 mg/mL FITC-BSA in PBS buffer for 2 h at 37 °C in dark surroundings. The stability of coated substrates was estimated by using LSCM after being rinsed with PBS buffer and water and dried with N2.