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Detailed study of BSA adsorption on micro- and nano-crystalline diamond/ #-SiC composite gradient films by time-resolved fluorescence microscopy Stephan Handschuh-Wang, Tao Wang, Sergey I. Druzhinin, Daniel Wesner, Xin Jiang, and Holger Schönherr Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b04177 • Publication Date (Web): 27 Dec 2016 Downloaded from http://pubs.acs.org on December 30, 2016
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Langmuir
Detailed study of BSA adsorption on micro- and nano-crystalline diamond/β-SiC composite gradient films by time-resolved fluorescence microscopy Stephan Handschuh-Wang,1,2,# Tao Wang,2,3,# Sergey I. Druzhinin,1,2 Daniel Wesner,1,2 Xin Jiang,2,3 * and Holger Schönherr1,2 * 1 Physical
Chemistry I, University of Siegen, Adolf-Reichwein-Str. 2, 57076 Siegen, Germany 2 Research Center of Micro and Nanochemistry and Engineering (Cµ), University of Siegen, Adolf-Reichwein-Str. 2, 57076 Siegen, Germany 3 Institute of Materials Engineering, University of Siegen, Paul-Bonatz-Str. 9-11, 57076 Siegen, Germany. #: Both authors contributed equally. ABSTRACT: The adsorption of bovine serum albumin (BSA) on micro- and nano-crystalline diamond/β-SiC composite films synthesized using the hot filament chemical vapor deposition (HFCVD) technique has been investigated by confocal fluorescence lifetime imaging microscopy. BSA labeled with fluorescein isothiocyanate (FITC) was employed as a probe. The BSAFITC-conjugate was found to preferentially adsorb on both O-/OH- terminated micro-crystalline and nano-crystalline diamond compared to the OH- terminated β-SiC, resulting in an increasing amount of BSA adsorbed to the gradient surfaces with increasing diamond/β-SiC ratio. The different strength of adsorption (>30 times for diamond with a grain size of 570 nm) coincides with different surface energy parameters and differing conformational changes upon adsorption. Fluorescence data of the adsorbed BSAFITC on the gradient film with different diamond coverage show a four-exponential decay with decay times of 3.71, 2.54, 0.66 and 0.13 ns for a grain size of 570 nm. The different decay times are attributed to fluorescence of thiourea fluorescein residuals of linked FITC distributed in BSA with different dye-dye and dye-surface distances. Fluorescence of BSAFITC undergoes an external dynamic fluorescence quenching on the diamond surface by H- and/or sp2-defects and/or by amorphous carbon or graphite phases. As the decay time was found to correlate linearly with the diamond grain size, an acceleration of the internal fluorescence concentration quenching because of structural changes of albumin due to adsorption, which also contributes to the quenching, is concluded to be a secondary contributor. These results suggest that the micro- and nano-crystalline diamond/β-SiC composite gradient films can be utilized to spatially control protein adsorption and diamond crystallite size, which facilitates systematic studies at these interesting (bio)interfaces.
posite films, which allow one to control to a certain extent the adsorption behavior of proteins on the surface.8 Due to the central role of conformational changes in protein adsorption and to better understand how bovine serum albumin interacts with the diamond/β-SiC composite films, we carried out the detailed time-resolved fluorescence microscopy study reported here. In general, various studies showed that surface chemistry and topography play a fundamental role in protein adsorption.9, 10 Künzler et al. reported that the surface roughness on micrometer and nanometer sized particles is an important surface parameter.11 It was also reported that the conformational changes of BSA adsorbed on gold nanoparticles depend on the particle size.12 Thus, the effects of roughness and crystallite size of diamond/β-SiC composite films on BSA adsorption need to be investigated. It has
1. INTRODUCTION The interaction of proteins and surfaces is a vitally important topic in biointerface science and beyond. Conformational changes in adsorbed protein are known to occur and can be attributed to strong protein-surface interaction.1, 2, 3, 4 Consequently, surface chemistry, but also surface topography were shown to play a fundamental role in protein adsorption to surfaces.2, 5 For applications in the biomaterials field, chemically stable materials that can be efficiently and robustly coated on surfaces are also of considerable interest.6, 7 We have reported previously on diamond and β-SiC films for biointerface applications.8 Due to different surface terminations of diamond and β-SiC, the surface chemistry could be controlled by synthesizing diamond/β-SiC com-
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further been suggested that conformational changes of adsorbed protein molecules can indirectly cause adverse reactions in the body, e.g. inflammation and thrombosis, after implantation of a foreign material/medical devices.13, 14 Recent studies have shown that platelets can adhere to adsorbed albumin, but only if the albumin undergoes more than a 34% loss in its α-helical structure, which is called a critical degree of unfolding.15 Therefore, conformational changes of adsorbed proteins play an important role in different organisms. In the literature various groups have utilized fluorescence spectroscopy, fluorescence microscopy and fluorescence lifetime imaging microscopy (FLIM), where data were analyzed in terms of average lifetime to study changes in protein conformation upon adsorption.14, 16, 17, 18, 19, 20, 21, 22 However, there have been thus far no studies of the conformational changes of BSA adsorbed on diamond films, SiC films or diamond/β-SiC composite films employing the fluorescence lifetimes of a probe molecule. In general, FLIM can be used to discriminate different fractions of a given fluorophore in different (nano)environments,21, 23 i.e. for determination of the morphology of aggregate.24 The fluorescence lifetime can change, among others, upon binding of a fluorophore to a biological target,25 and due to surface effects.26 Excited state lifetime and quenching effects of fluorescein isothiocyanate (FITC) on surfaces were investigated by Martini et al.26 On gold surfaces the average lifetime and quantum yield of the aminofluorescein fluorophore remained virtually constant (with an average lifetime between 3.6 ± 0.1 and 4.1 ± 0.1 ns), when inter dye distance on the gold surface decreases, whereas for bare silica particle the excited state lifetime decreased with lowering inter dye distance (with an average lifetime ranging from 3.4 ns for 10.1 nm inter dye distance to 2.2 ns for an inter dye distance of 4 nm). The effect of the shortening of the excited state lifetime was ascribed to dye dimerization and energy transfer from the monomer toward these H-type dimers, whereas the presence of at least two lifetime components was ascribed to the heterogeneous distribution of the dye molecules onto the surface.26 In the study of Hungerford et al.25 FITC was used to label BSA. Their conjugate (BSAFITC) exhibited at low labeling ratios already two distinct excited state lifetimes, namely τ1 ≈ 4 ns and τ2 ≈ 0.6 ns. By increasing the labeling ratio, a third intermediate excited state lifetime τ3 ≈1.5 ns arose, while the other excited state lifetimes decreased. FITC has also proven to be useful to study BSA packing and conformational changes upon adsorption onto polycaprolactone surfaces by fluorescence lifetime imaging.16 The average fluorescence lifetime decreased with reduced average distance between FITC residuals in BSA according to Förster resonance energy transfer (FRET).25 This can be utilized to measure intermolecular distance between the FITC labels.16, 27 In this context, FLIM was used here to monitor possible conformational changes of BSA upon adsorption.28, 29, 30 In our recent publication8 we utilized BSAFITC as a probe for the preferential adsorption of BSA on diamond/β-SiC gradient films, but further analysis of the excited state lifetimes was not conducted. Here FLIM data were employed
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to evaluate the interactions between BSAFITC and the diamond/β-SiC composite surfaces. In particular, the excited state lifetimes were analyzed as a measure for conformational changes of BSA adsorbed on β-SiC, diamond, and diamond/β-SiC composite surfaces with different surface concentration and different size of diamond crystallites.
2. EXPERIMENTAL PART Materials. H2SO4 (ACS reagent), H2O2 (30 wt.% in H2O, ACS reagent), HF (> 48 % in H2O, ACS reagent), tetramethylsilane (≥ 99.9%) and KNO3 (p.a., ≥ 65%) were purchased from Sigma-Aldrich. Tris buffer was prepared by dissolving 0.01 mol Tris (tris(hydroxymethyl) aminomethane, ≥ 99%, Sigma-Aldrich) in 1 L Milli-Q water (drawn from a Millipore Direct Q8 system (Millipore, Schwalbach, with Millimark Express 40 filter, Merck, Germany) with a resistivity of 18.0 MΩ cm.). Afterwards, the pH was adjusted to 7 by addition of HCl. Film Deposition. Diamond/β-SiC composite gradient films were synthesized by hot-filament chemical vapor deposition (HFCVD) with a special filament/substrate configuration (self-built8) on P-type (100) Si wafers (Siegert Consulting e. K. Aachen, German), as described in an earlier paper.8 Prior to film deposition, the substrates were immersed in piranha solution (H2SO4:H2O2 3:1) for 30 min followed by ultrasound cleaning in distilled water for three times. Afterwards the samples were ultrasonically seeded with a 5 nm nanodiamond dispersion (0.05 wt% in water) for 30 min in order to enhance the diamond nucleation.31 The samples were then dried in stream of N2. The deposition was carried out at a constant gas pressure of 30 mbar. The flow rates of H2, CH4 and tetramethylsilane (TMS, 1% TMS diluted in H2) were maintained at 500, 5 and 30 sccm. The filament temperature was around 2500°C. The parameters for deposition of micro- and nanocrystalline composite films were the same except for the substrate temperature. For microcrystalline composite films (diamond grain size >300 nm), the substrate temperature was kept between 883°C (± 20°C) and 762°C (± 20°C) along the length of substrate. For nanocrystalline composite films (diamond grain size
