Supporting Information Material

Supporting Information Material

Plasma Protein Adsorption to Zwitterionic Poly (Carboxybetaine Methacrylate) Modified Surfaces: Chain Chemistry and End-group Effects on Protein Adsorption Kinetics, Adsorbed Amounts and Immunoblots

Sinoj Abraham1,2, Markian S. Bahniuk3 and Larry D. Unsworth1-3*

1Chemical and Materials Engineering Department - University of Alberta; 2National Research Council (Canada) - National Institute for Nanotechnology; 3Biomedical Engineering Department-University of Alberta; Edmonton, Alberta, Canada

E-mail:

Experimental

Surface functionalization:The nitroxide radical and brominated trimethoxysilane (BrTMOS) was allowed to react using copper(I)bromide-pentamethyldiethylenetriamine (CuBr-PMDETA) catalyst in anhydrous toluene media to form corresponding -phosphonate alkoxyamine. Oxidized silica wafer substrates were functionalized with these alkoxyamines by immersed in their toluene solution (0.5g in 20mL) for 3h at 50 OC. They were then removed, washed and kept in a vacuum oven for 5h at 80OC. Surface initiated nitroxide mediated free radical polymerization (NMFRP) was performed on these substrates with CBMA monomers [1]. The surface graft densities on these substrates were determined by thermo-gravimetric analysis and the surface compositions were determined by XPS. The surface hydrophilicity was obtained by measuring the advancing and receeding water contact angle measurements. The molecular weight of polymers was determined using GPC analysis [1].

In Situ Ellipsometry: Characterization of Initiator/Polymer Modified Surfaces: Silica wafers were thermally oxidized (900 OC for 30 min) to form the SiO2 layer and was characterized prior to initiator/polymer functionalization. The refractive index of silica was taken as 1.456 at = 632.8 nm [2] and its oxide layer thickness was measured to be 20 ± 2 nm using the optical properties (n = 3.877, k = 0.016). Ellipsometry data were fitted with multilayer models using the WVASE 32 software and a Cauchy model (A = 1.45, B = 0.01 and C = 0). Increase in thickness up to 1.5 ± 0.5 nm was observed after grafting alkoxyamineinitiators on these surfaces [1]. The refractive index of all PCBMA layers was fixed at the value of 1.5 for the analysis [3].

In Situ Ellipsometry: Characterization of Single Protein Adsorption: A 10mM phosphate buffer (PB, pH=7) was prepared from disodium phosphate and potassium phosphate and used in the preparation of all protein samples. Protein solutions were prepared with a final concentration of 1, 0.75, 0.5 and 0.25 mg/mL. Solution protein concentrations were verified using a DU 730 Life Sciences UV/Vis spectrophotometer (Beckman Coulter) by measuring the absorbance at 280nm and with molar extinction coefficient of each protein, (ie. Lys = 36,000 M-1cm-1, -La = 28,175 M-1cm-1, HSA = 36600 M-1cm-1, Fbn = 51500 M-1cm-1). These solutions were then used for all ellipsometry experiments.

In situ ellipsometry experiments were conducted as generally outlined above, where phosphate buffer (PB) was injected into the cell containing the pre-characterized initiator or polymer modified surface and the background measurements obtained for 15 min. Protein solutions were then injected into the cell and the adsorption was monitored for 2 hrs, then rinsed with copious amounts of PB. Measurements were stopped until a negligible change in the signal was noticed and were repeated thrice for all surfaces with each protein. The liquid cell was washed with detergent (Alconox) and deionized water between experimental trials to remove any residual proteins. All the measurements and the storage of protein solutions were done at room temperature to avoid temperature induce birefringence in the quartz windows that might lead to changes in the  values of ellipsometric data. The thickness of adsorbate film was fit to the experimental data as function of time, using Cauchy parameters A = 1.46 (Lys), 1.46 (-La), 1.58 (HSA), 1.59 (Fbn) and constant values for B = 0.01and C=0 for protein layers. Cauchy dispersion model can be used to describe the dispersion of the refractive index of transparent materials using the Cauchy equation n() = A + B/2 + C/4 , where is the wavelength and A, B, and C are the fit parameters [4]. Using De Feijter’s equation, the mass of adsorbate film was calculated using the thickness and refractive index [5].Refractive index of the protein in solution is assumed to be a linear function of its concentration, the absolute amount  of adsorbed protein can be determined using:

where df and nf are the thickness and refractive index of the adsorbed film, respectively, na is the refractive index of the aqueous medium (1.357 for PBS buffer), and dn/dc is the increment of refractive index of the protein solution versus its concentration, which is approximated to 0.183 ± 0.005 ml/g [6,7]. Non-linear regression analysis of the adsorption model was performed to determine the kinetics of protein adsorption by using commercial software of Sigmaplot version11 via the Marquardt–Levenberg algorithm.

Table S1. Primary antibodies used for immunoblotting of plasma proteins adsorbed to PCBMA Surfaces

a Horseradish Peroxidase Conjugate

GPC Chromatograms for Mn and PDI determination:

Figure S1.GPC profiles of [poly(carboxybetaine methacrylamide)] with phosphonate initiator.

Figure S2.GPC profiles of [poly(carboxybetaine methacrylamide)] with TEMPO Initiators.

TGA: Representative dataset

Figure S3.RepresentativeThermo gravimetric analysis of initiator and polymer (PolyCBMA-5) bonded silicon wafer surface. The grafting density was calculated on the basis of TGA weight loss.

XPS:Representative dataset

Figure S4.RepresentativeXPS profiles of PolyCBMA-5 (a) with TEMPO and (b) with Phospho end-groups.

References

[1]S. Abraham, L.D. Unsworth, J. Polym. Sci. Part A: Polym. Chem. 49 (2011) 1051.

[2]D.R. Lide (Eds.), CRC Handbook of Chemistry and Physics, 85th Ed. CRC Press, 2004, Chapter 1.

[3]E. Kharlampieva, V.A. Izumrudov, S.A. Sukhishvili, Macromolecules 40 (2007) 3663.

[4]H.G. Tompkins, W.A. McGahan (Eds.) Spectroscopic Ellipsometry and Reflectometry A User’s Guide, NewYork, Wiley, 1999, 28.

[5]J.A. De Feijter, J. Benjamins, F.A. Veer, Biopolymers 17 (1978) 1759.

[6]V. Ball, J.J. Ramsden, Biopolymers 46 (1998) 489.

[7]L.D. Unsworth, Z. Tun, H. Sheardown, J.L. Brash, J. Colloid. Interface. Sci. 296 (2006) 520.