Nonfouling poly(ethylene oxide) layers end-tethered to polydopamine.

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 hetero-bifunctional 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.

Morphological and molecular analysis of the Nematostella vectensis cnidom.

The starlet sea anemone Nematostella vectensis is an emerging model organism for developmental and evolutionary biology. Due to the availability of genome data and its amenability to genetic manipulation Nematostella serves as a source for comparative molecular and phylogenetic studies. Despite this fact, the characterization of the nematocyst inventory and of nematocyst-specific genes is still fragmentary and sometimes misleading in this cnidarian species. Here, we present a thorough qualitative and quantitative analysis of nematocysts in Nematostella vectensis. In addition, we have cloned major nematocyst components, Nematostella minicollagens 1, 3 and 4, and show their expression patterns by in situ hybridization and immunocytochemistry using specific antibodies. Our data provides tools and insights for further studies on nematocyst morphogenesis in Nematostella and comparative evolution in cnidarians.

In vitro observation of dynamic ordering processes in the extracellular matrix of living, adherent cells.

Collecting information at the interface between living cells and artificial substrates is exceedingly difficult. The extracellular matrix (ECM) mediates all cell-substrate interactions, and its ordered, fibrillar constituents are organized with nanometer precision. The proceedings at this interface are highly dynamic and delicate. In order to understand factors governing biocompatibility or its counterpart antifouling, it is necessary to probe this interface without disrupting labels or fixation and with sufficient temporal resolution. Here the authors combine nonlinear optical spectroscopy (sum-frequency-generation) and microscopy (second-harmonic-generation), fluorescence microscopy, and quartz crystal microgravimetry with dissipation monitoring in a strategy to elucidate molecular ordering processes in the ECM of living cells. Artificially (fibronectin and collagen I) and naturally ordered ECM fibrils (zebrafish, Danio rerio) were subjected to nonlinear optical analysis and were found to be clearly distinguishable from the background signals of diffusive proteins in the ECM. The initial steps of fibril deposition and ordering were observed in vitro as early as 1 h after cell seeding. The ability to follow the first steps of cell-substrate interactions in spite of the low amount of material present at this interface is expected to prove useful for the assessment of biomedical and environmental interfaces.

Probing the extracellular matrix with sum-frequency-generation spectroscopy.

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.