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.