Nonfouling surfaces capable of reducing protein adsorption are highly desirable in a wide range of applications. Coating of surfaces with poly(ethylene oxide) (PEO), a water-soluble, nontoxic, and nonimmunogenic polymer, is most frequently used to reduce nonspecific protein adsorption. Here we show how to prepare dense PEO brushes on virtually any substrate by tethering PEO to polydopamine (PDA)-modified surfaces. The chain lengths of heterobifunctional PEOs were varied in the range of 45−500 oxyethylene units (M n = 2000−20 000). End-tethering of PEO chains was performed through amine and thiol headgroups from reactive polymer melts to minimize excluded volume effects. Surface plasmon resonance (SPR) was applied to investigate the adsorption of model protein solutions and complex biologic medium (human blood plasma) to the densely packed PEO brushes. The level of protein adsorption of human serum albumin and fibrinogen solutions was below the detection limit of the SPR measurements for all PEO chains end-tethered to PDA, thus exceeding the protein resistance of PEO layers tethered directly on gold. It was found that the surface resistance to adsorption of lysozyme and human blood plasma increased with increasing length and brush character of the PEO chains end-tethered to PDA with a similar or better resistance in comparison to PEO layers on gold. Furthermore, the chain density, thickness, swelling, and conformation of PEO layers were determined using spectroscopic ellipsometry (SE), dynamic water contact angle (DCA) measurements, infrared reflection− absorption spectroscopy (IRRAS), and vibrational sum-frequency-generation (VSFG) spectroscopy, the latter in air and water.

Fixed fibronectin-coated gold surfaces with and without adherent embryonic fibroblasts were probed via vibrational sum-frequency-generation (SFG) spectroscopy. The SFG spectra were compared to infrared reflection-absorption spectroscopy (IRRAS) data in the CH stretching region. Noticeable differences were observed in the IRRAS spectra of the samples, whereas SFG spectra of the same samples were largely similar. These results suggest that cells with their overall random distribution of CH groups do not contribute to the SFG spectra, resulting in similar spectral features related to the fibronectin coating regardless of whether cells are adhered to it. Furthermore, SFG spectra of cells adhered directly on gold were found to have features similar to those of cells adhered on fibronectin-covered gold. Additional experiments with living cells treated in vitro with the high-powered lasers used in these experiments did not result in any visible radiation damage to the cells. These results demonstrate the feasibility of using SFG spectroscopy as an experimental tool to characterize the extracellular matrix (ECM) layer adjacent to a gold substrate beneath a layer of cells and also suggest that this technique could be operated to examine the ECM in vitro.

The ability to probe an interface beneath a layer of living cells in situ without the need for labeling and fixation has the potential to unlock some of the key questions in cell biology and biointerfacial phenomena. Here, we show that vibrational sum frequency generation (SFG) spectroscopy can be used to detect alkanethiol self-assembled monolayers (SAMs) buried underneath a layer of living erythrocytes (ECs). SFG spectra with and without ECs showed the spectral signatures typical of these SAMs, indicating that the signal was being generated solely by the SAM and was not influenced by the presence of cells. Direct comparison of infrared spectroscopy to SFG measurements of cells adhered on a fibronectin layer showed that the SFG signal emanated solely from this layer. These results have important implications for the characterization of surfaces in biomedical, environmental, and industrial applications.