Current Published Papers

Freeman, N. J., Peel, Swann, M.J., Cross, G.H., Reeves, A., Brand S., Lu, J.R. Real Time, High Resolution Studies of Protein Adsorption and Structure at the Solid-Liquid Interface Using Dual Polarisation Interferometry. J. Phys.: Condens. Matter, (2004), 16 S2493-S2496.

Abstract: A novel method for the analysis of thin biological films, called dual polarization interferometry (DPI), is described. This high resolution (<1 Å), laboratory based technique allows the thickness and refractive index (density) of biological molecules adsorbing or reacting at the solid–liquid interface to be measured in real time (up to 10 measurements per second). Results from the adsorption of bovine serum albumin (BSA) on to a silicon oxynitride chip surface are presented to demonstrate how time dependent molecular behaviour can be examined using DPI. Mechanistic and structural information relating to the adsorption process is obtained as a function of the solution pH.

Armstrong, J., Salacinski, H.J., Mu, Q., Seifalian, A. M., Peel, L., Freeman, N., Holt, C.M, Lu J.R. Interfacial Adsorption of Fibrinogen and its Inhibition by RGD Peptide: A Combined Physical Study. J. Phys.: Condens. Matter, (2004), 16 S2483-S2491.

Abstract: The Arg–Gly–Asp (RGD) peptide sequence is known as a cell recognition site for numerous adhesive proteins present in the extracellular matrix (ECM) and in blood. Whilst surface immobilized RGD groups enhance cell attachment, RGD components present in solution can effectively inhibit cell attachment by competing with endogenous ligands for the same recognition site. In contrast to the widely reported binding to cell integrin, this study demonstrates a new RGD feature: its inhibitive effect on fibrinogen adsorption. Through a combined analysis of spectroscopic ellipsometry, neutron reflection and dual polarization interferometry, we show that the kinetic process of fibrinogen adsorption as a model pro-coagulant at the silica/solution interface and in the absence of any cells can be substantially reduced by the addition of RGD in solution and that the extent of the reduction is dependent on the relative concentration of RGD.

Lu, J.R., Swann, M. J., Peel, L.L., Freeman, N., J. Lysozyme Adsorption Studies at the Silica-Water Interface using Dual Polarisation Interferometry. Langmuir, (2004), 20 1827-1832.

Abstract: Lysozyme adsorption at the silica/water interface has been studied using a new analytical technique called dual polarization interferometry. This laboratory-based technique allows the build up or removal of molecular layers adsorbing or reacting on a lightly doped silicon dioxide (silica) surface to be measured in terms of thickness and refractive index changes with time. Lysozyme adsorption was studied at a range of concentrations from 0.03 to 4.0 g dm ^-3 and at both pH 4 and pH 7. Adsorbed layers ranging from 14 to 43 ± 1 Å in thickness and 0.21 to 2.36 ( 0.05 mg m ^-2 in mass coverage were observed at pH 4 with increasing lysozyme concentration, indicating a strong deformation of the monolayer over the low concentration range and the formation of an almost complete sideways-on bilayer toward the high concentration of 4 g dm-3. At pH 7, the thickness of adsorbed layers varied from 16 to 54 ±1 Å with significantly higher surface coverage (0.74 to 3.29±0.05 mgm ^-2 ), again indicating structural deformation during the initial monolayer formation, followed by a gradual transition to bilayer adsorption over the high concentration end. The pH recycling performed at a fixed lysozyme concentration of 1.0 g dm-3 indicated a broadly reversible adsorption regardless of whether the pH was cycled from pH 7 to pH 4 and back again or vice versa. These observations are in good agreement with earlier studies undertaken using neutron reflection although the fine details of molecular orientations in the layers differ subtly.

Swann, M. J., Peel, L.L., Carrington, S., Freeman N. J., Dual Polarisation Interferometry: An Analytical Technique to Measure Changes in Protein Structure in Real Time, to Determine the Stoichiometry of Binding Events and to Differentiate Between Specific and Non-Specific Interaction. Analytical Biochemistry, (2004), 329 190-198.

Abstract: The study of solution-phase interactions between small molecules and immobilized proteins is of intense interest, especially to the pharmaceutical industry. An optical sensing technique, dual polarization interferometry, has been employed for the detailed study of a model protein system, namely, D-biotin interactions with streptavidin immobilized on a solid surface. Changes in thickness and density of an immobilized streptavidin layer as a result of the binding of D-biotin have been directly measured in solution and in real time. The results obtained from this approach are in excellent agreement with X-ray crystallographic data for the structural changes expected in the streptavidin-D-biotin system. The mass changes measured on binding D-biotin also agree closely with anticipated binding capacity values. Determination of the density changes occurring in the protein adlayer provides a means for differentiation between specific and nonspecific interactions.

Oikawa, T., Yamaguchi, H., Itoh, T., Kato, M., Ijuin, T., Yamazaki, D., Suetsugu S., Takenawa T., PtdIns(3,4,5) P3 Binding is Necessary for WAVE2-induced Formation of Lamellipodia. Nature Cell Biology, (2004), 6 420-426.

Abstract: Polarized cell movement is triggered by the development of a PtdIns(3,4,5)P3 gradient at the membrane, which is followed by rearrangement of the actin cytoskeleton. The WASP family verprolin homologous protein (WAVE) is essential for lamellipodium formation at the leading edge by activating the Arp2/3 complex downstream of Rac GTPase. Here, we report that WAVE2 binds to PtdIns(3,4,5)P3 through its basic domain. The amino-terminal portion of WAVE2, which includes the PtdIns(3,4,5)P3-binding sequence, was localized at the leading edge of lamellipodia induced by an active form of Rac (RacDA) or by treatment with platelet derived growth factor (PDGF). Production of PtdIns(3,4,5)P3 at the cell membrane by myristoylated phosphatidylinositol-3-OH kinase (PI(3)K) is sufficient to recruit WAVE2 in the presence of dominant-negative Rac and latrunculin, demonstrating that PtdIns(3,4,5)P3 alone is able to recruit WAVE2. Expression of a full-length mutant of WAVE2 that lacks the lipid-binding activity inhibited proper formation of lamellipodia induced by RacDA. These results suggest that one of the products of PI(3)K, PtdIns(3,4,5)P3, recruits WAVE2 to the polarized membrane and that this recruitment is essential for lamellipodium formation at the leading edge.

Cross, G. H., Reeves, A., Brand, S., Swann, M. J., Peel, L., Freeman, N.J., Lu, J.R., The Metrics of Surface Adsorbed Small Molecules on the Young’s Fringe Dual-Slab Waveguide Interferometer. J. Phys. D: Appl. Phys., (2004), 37 74-80.

Abstract: A method for analysing thin films using a dual-waveguide interferometric technique is described. Alternate dual polarization addressing of the interferometer sensor using a ferroelectric liquid crystal polarization switch allowed the opto-geometrical properties (density and thickness) of adsorbed layers at a solid–liquid interface to be determined. Differences in the waveguide mode dispersion between the transverse electric and transverse magnetic modes allowed unique combinations of layer thickness and refractive index to be determined at all stages of the layer formation process. The technique has been verified by comparing the analysis of the surface adsorption of surfactants with data obtained using neutron scattering techniques, observing their behaviour on trimethylsilane coated silicon oxynitride surfaces. The data obtained were found to be in excellent agreement with analogous neutron scattering experiments and the precision of the measurements taken to be of the order of 40 pm with respect to adsorbed layer thicknesses. The study was extended to a series of surfactants whose layer morphology could be correlated with their hydrophilicity/lipophilicity balance. Those in the series with longer alkyl chains were observed to form thinner, denser layers at the hydrophobic solid/aqueous liquid interface and the degree of order attained at sub-critical micelle concentrations to be correlated with molecular fluidity. The technique is expected to find utility with those interested in thin film analysis. An important and growing area of application is within the life sciences, especially in the field of protein structure and function.

Brennan, D., O’Brien, P., O’Brien, J., Freeman, N., Swann, M. Development and test of an integrated Microsystem for HPLC separation and detection using Refractive index measurements. Sensors & Actuators, (2004),103 184-189.

Keywords: Micro-fluidics; Micro-HPLC; Packaging; Refractive index

Abstract: In recent years optical waveguide sensors have been used for applications as biosensors and chemical sensors. Often the fluidic element of the sensor has a large working volume (_l) and full device integration is not undertaken. We look at a number of fabrication routes available to integrate microfluidic and optical detection components to achieve a fully integrated microsystem and highlight some of the technical issues arising in different integration strategies. Wafer and chip integration strategies using anodic, thin film and polymer bonding for the fluidic module and the optical sensor were investigated. The integrated microsystem was evaluated for fluidic and optical performance, integration showed no significant impact on performance of either component. The fluidic system presents a nanolitre detection volume offering reduced sample volumes in HPLC applications. Initial optical performance of the integrated structure was carried out using a refractive index measurement system, the Farfield Analight®Bio250.

Reeves, A., Theoretical Studies of One-dimensional and Two-dimensional Photonic Structures. PhD Thesis, University of Durham, (2004).

Freeman, N., From microstructures to measurements in the pharmaceutical industry. The IEE Seminar on MNT in Medicine, (2004), 2004/10743, 79-92.