Letter Cite This: ACS Macro Lett. 2018, 7, 498−503
pubs.acs.org/macroletters
Grafting Density Impacts Local Nanoscale Hydrophobicity in Poly(ethylene glycol) Brushes David Faulón Marruecos, Daniel F. Kienle, Joel L. Kaar,* and Daniel K. Schwartz* Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, Colorado 80309, United States S Supporting Information *
ABSTRACT: Accumulated single-molecule observations of a fluorescent solvatochromic probe molecule were found to provide detailed local information about nanoscale hydrophobicity in polymer brushes. Using this approach, we showed that local hydrophobicity in poly(ethylene glycol) (PEG) brushes was spatially heterogeneous and increased with the surface grafting density of the polymer chains. These findings may provide an explanation for prior observations of the denaturation of surface-adsorbed proteins on PEG brushes with high grafting densities, which is believed to influence protein-mediated cell−surface interactions. Moreover, by employing the broad range of existing environmentally sensitive fluorophores, this approach may potentially be used to characterize nanoscale changes in a variety of physicochemical properties within polymeric materials.
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hydrophilicity of that surface. The explicit resolution of this issue has remained elusive due to the lack of available experimental methods to detect nanoscale changes in hydrophobicity within the brush layer. Using conventional methods for measuring surface hydrophobicity, such subtle and local changes are typically masked by the ensemble-averaging nature of such measurements on surfaces that are intrinsically heterogeneous. For example, the effect of PEG brush density on the static contact angle of water is empirically undetectable. Additionally, traditional methods are insensitive to temporal fluctuations in the existence of nanoscale hydrophobic niches due to dynamic changes in the conformation of the polymer chains. Although sum frequency generation spectroscopy may be used to characterize the in situ hydration of polymer brushes, as well as provide information about the interaction of water molecules with the polymer chains, the information provided by this technique is also ensemble-averaged.8,21 Here, we developed and employed a new method to investigate the impact of grafting density on the local hydration of PEG brushes via super-resolution fluorescence mapping. This approach, which was based on mapping using accumulated probe trajectories (MAPT),22−24 exploited the solvatochomic properties of the environmentally sensitive fluorophore nitrobenzoxadiazole (NBD). Notably, the fluorescent emission of the NBD derivative 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid (NBD-X) undergoes a dramatic red-shift and has reduced intensity in a hydrophilic (polar) environment compared with the emission in a hydrophobic (nonpolar) environment.25,26 Changes in the emission wavelength of NBD-
olymer brushes composed of poly(ethylene glycol) (PEG) are widely used as a steric barrier to inhibit the adsorption of proteins to materials in biological milieu.1−8 However, while PEG brushes often reduce protein adsorption, they do not eliminate it entirely, and the adsorption of proteins to PEG brushes depends on the grafting density (σ) of PEG as well as the molecular weight (MW) of the PEG chains.9−18 In particular, for a given PEG MW, there is often an apparent optimum “protein-resistant” range of grafting density outside of which protein accumulation increases.9,10,16−19 While the molecular basis for this optimum is unclear, it has been hypothesized to arise from the emergence of nanoscale hydrophobic regions in the brush at higher grafting density. Unfolded protein molecules may have strong affinity to such regions, which could enhance surface-mediated protein aggregation and increase adsorption. We recently investigated the relationship between grafting density and protein adsorption and unfolding on PEG brushes using single-molecule (SM) Förster resonance energy transfer methods that are uniquely sensitive to interfacial dynamics and conformational changes.20 Interestingly, while brushes with higher grafting density decreased the overall rate of protein adsorption, they also stabilized unfolded protein molecules and increased the rate of protein unfolding, leading to a greater fraction of accumulated unfolded protein. Since unfolded protein molecules are presumably stabilized on hydrophobic sites, these findings support the hypothetical connection between grafting density and a decrease in the local hydration within the brush layer. While some findings have suggested that dense PEG brushes may exhibit an increased hydrophobic character,10,15,17 this counters the more conventional belief that increasing the amount of PEG on a given surface also increases the overall © XXXX American Chemical Society
Received: January 2, 2018 Accepted: April 2, 2018
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DOI: 10.1021/acsmacrolett.8b00004 ACS Macro Lett. 2018, 7, 498−503
Letter
ACS Macro Letters X were mapped molecule-by-molecule with nanoscale resolution on PEG brushes with low (0.16 chains/nm2) and high (0.34 chains/nm2) grafting density. Super-resolution maps of these changes at the brush−liquid interface were generated using SM total internal reflection fluorescence (TIRF) microscopy of the brush surfaces during exposure to solution containing NBD-X (Figure 1a). The resulting maps provided new insights into the increase in nanoscale hydrophobic sites with grafting density, which is critical for tuning the interaction between protein and PEG brushes and, in turn, modulating cell responses to denatured protein on the brush surface.27−31 The nanoscale mapping of surface hydrophobicity via the spectral shift of NBD-X was validated by characterizing nanopatterned surfaces comprising hydrophilic fused silica (FS) and hydrophobic trimethylsiloxane (TMS) domains. Dual-channel images were acquired by splitting the fluorescent emission of NBD-X into spectral channels with peak transmission wavelengths at 529 and 560 nm, respectively (Figure 1b, left and middle panels). The positions and dual channel intensities of individual NBD-X molecules from each sequential image were identified and tracked using custom software. Detailed descriptions of the surface preparation, experimental apparatus, and software algorithms are provided in the Supporting Information. The intensities from each channel corresponding to individual objects were used to define a “Hydrophobicity Index” (HI) as ⎛I ⎞ Hydrophobicity Index = log10⎜ 529 ⎟ ⎝ I560 ⎠
(1)
where I529 was the object intensity measured in the 529 nm channel and I560 was the object intensity measured in the 560 nm channel. Larger values of HI corresponded to a more hydrophobic environment while an HI value of zero represented a site with similar hydrophobic and hydrophilic character. As such, sites that had an HI value of zero were amphiphilic in nature, where the hydrophobicity and hydrophilicity of the site was equally balanced. This allowed us to characterize the local chemical environment surrounding each probe molecule with resolution limited only by the localization precision (70 ± 30 nm). Super-resolution surface maps of HI were created by binning all objects by spatial position. For this, we divided the field of view into an array of 217 × 217 nm2 bins. The HI bin values were calculated by averaging the HI values of all objects that occupied a bin throughout the movie. A spatial map of HI, along with a probability density of the data corresponding to the FS and TMS regions, is shown in Figure 1b (right panel) and c, respectively. As expected, the objects in the TMS regions had HI values that were systematically larger than on the FS regions. Interestingly, a small hydrophobic region was observed at the center of each FS square, suggesting that the TMS layer was not fully degraded during photopatterning. The same measurement and analytical methods were applied to PEG polymer brushes with low and high grafting densities, which were made by using a grafting-to approach with a good and a poor solvent, respectively32 (see the Supporting Information for details of brush preparation). The brushes were characterized by ellipsometry in the dry state, and the thickness was converted to a grafting density as described previously.20
Figure 1. (a) Experimental schematic. The excitation laser is totally internally reflected at the silica−water interface, creating an evanescent field that excites NBD-X. Upon adsorption on hydrophilic (FS) patches, the dye emits at a longer wavelength than on hydrophobic (TMS) patches. Emitted photons are spectrally separated and steered to different regions of a camera sensor. (b) Super-resolution map of a patterned surface, with FS squares surrounded by TMS regions, 499
DOI: 10.1021/acsmacrolett.8b00004 ACS Macro Lett. 2018, 7, 498−503
Letter
ACS Macro Letters Figure 1. continued generated from 2 × 106 NBD-X trajectories. The color scale represents changes in local hydrophobicity based on the parameter HI, as described in the text. (c) Probability density distributions of HI values. An image of the time-averaged HI values was used to define regions corresponding to FS and TMS. The HI values from these regions were used to generate the corresponding probability density distributions. The p-value of the Student t test on the mean of the distributions was
