Quantification of surface-bound proteins by fluorometric assay: Comparison with quartz crystal microbalance and amido black assay

Protein adsorption is of major and widespread interest, being useful in the fundamental understanding of biological processes at interfaces through to the development of new materials. A number of techniques are commonly used to study protein adhesion, but few are directly quantitative. Here we describe the use of Nano Orange, a fluorometric assay, to quantitatively assess the adsorption of bovine fibrinogen and albumin onto model hydrophilic (OH terminated) and hydrophobic (CH3 terminated) surfaces. Results obtained using this method allowed the calibration of previously unquantifiable data obtained on the same surfaces using quartz crystal microbalance measurements and an amido black protein assay. Both proteins were found to adsorb with higher affinity but with lower saturation levels onto hydrophobic surfaces. All three analytical techniques showed similar trends in binding strength and relative amounts adsorbed over a range of protein concentrations, although the fluorometric analysis was the only method to give absolute quantities of surface-bound protein. The versatility of the fluorometric assay was also probed by analyzing protein adsorption onto porous superhydrophobic and superhydrophilic surfaces. Results obtained using the assay in conjunction with these surfaces were surface chemistry dependent. Imbibition of water into the superhydrophilic coatings provided greater surface area for protein adsorption, although the protein surface density was less than that found on a comparable flat hydrophilic surface. Superhydrophobic surfaces prevented protein solution penetration. This paper demonstrates the potential of a fluorometric assay to be used as an external calibration for other techniques following protein adsorption processes or as a supplemental method to study protein adsorption. Differences in protein adsorption onto hydrophilic vs superhydrophilic and hydrophobic vs superhydrophobic surfaces are highlighted.     

In situ data collection and structure refinement from microcapillary protein crystallization

In situ X-ray data collection has the potential to eliminate the challenging task of mounting and cryocooling often fragile protein crystals, reducing a major bottleneck in the structure determination process. An apparatus used to grow protein crystals in capillaries and to compare the background X-ray scattering of the components, including thin-walled glass capillaries against Teflon, and various fluorocarbon oils against each other, is described. Using thaumatin as a test case at 1.8 ? resolution, this study demonstrates that high-resolution electron density maps and refined models can be obtained from in situ diffraction of crystals grown in microcapillaries.     

Multivariate analysis of emission decay matrices for distinguishing ground state heterogeneity and excited state reactions of tryptophan

The amino acid tryptophan displays emission solvatochromism, an emission maximum that shifts with solvent polarity, which is often used in protein studies to indicate local environment hydrophobicity. Use of tryptophan solvatochromism in time-resolved protein studies has traditionally been complicated due to the undescribed photokinetics that result in a characteristic multiexponential emission decay. For the first time, by application of the photokinetic matrix decomposition (PMD) multivariate curve resolution method to time-resolved emission decay (TRED) data, a distinguishment between ground state heterogeneous (GSH) and excited state reaction (ESR) type photokinetics of tryptophan in solution is made possible. It is found that molecular tryptophan displays two emission spectra that decay independently, suggesting GSH type photokinetics, one at 347 nm with a lifetime of 0.5 ns and one at 363 nm with a lifetime of 3.1 ns. When tryptophan is incorporated into a peptide, mastoparan X, the data similarly contain two emission spectra that decay independently, but are shifted in wavelength. Photobleaching experiments confirm that the PMD method is sensitive to tryptophan emission quenching, and therefore may be applied to determine the photokinetics of tryptophan that occur in proteins. Future applications of PMD analysis of tryptophan TRED data as a bioanalytical tool for further characterizing dynamic protein processes are discussed.     

Properties, isomorphisms and enumeration of 2-Quasi-Magic Sudoku grids

A Sudoku grid is a 9×9 Latin square further constrained to have nine non-overlapping 3×3 mini-grids each of which contains the values 1–9. In Δ-Quasi-Magic Sudoku a further constraint is imposed such that every row, column and diagonal in each mini-grid sums to an integer in the interval [15−Δ,15+Δ]. The problem of proving certain (computationally known) results for Δ=2 concerning mini-grids and bands (rows of mini-grids) was posed at the British Combinatorial Conference in 2007. These proofs are presented and extensions of these provide a full combinatorial enumeration for the total number of completed 2-Quasi-Magic Sudoku grids. It is also shown that there are 40 isomorphism classes of completed 2-Quasi-Magic Sudoku grids.     

Tuning the adhesion of layer-by-layer films to the physicochemical properties of inner limiting membranes using nanoparticles

Retinal trauma is a serious concern for patients undergoing inner limiting membrane (ILM) peeling to correct for various vitreoretinal interface conditions. This mechanical trauma can be prevented by modifying the surface of surgical instruments to increase adhesion to the ILM. To this effect, we have studied the effects of roughness and surface charge on the adhesive properties of ILMs by utilizing layer-by-layer (LbL) films with embedded gold nanoparticles (LbL-AuNP films). LbL films were assembled on atomic force microscopy (AFM) tipless cantilevers. Topographical analysis of these films, with and without nanoparticles, showed that LbL films with nanoparticles had a higher rms roughness compared to films alone or unmodified cantilevers. Nanoparticle-modified LbL films significantly increased the adhesion forces at the cantilever-ILM interface, compared to LbL films without particles. Surprisingly, adsorption of gold nanoparticles onto the AFM cantilevers caused increases in adhesion forces greater than those measured with LbL-AuNP films. These results have important implications for the design of surface modifications for vitreoretinal surgical instruments.     

Anomalous Luminescence of Some Organic Solvents

Luminescence from some organic solvents has been observed when irradiated by infrared (1060nm) laser light. This effect is unusual in that the luminescence occurs in the visible spectrum while the excitation is in the infrared. Furthermore, a precondition is required for such an effect to occur, i.e. the sample must first be broken down (creation of a spark in the sample at the focuss of the laser) permanent.