Current Published Papers

Furutania, M., Tsujitaa, K., Itoha, T., Ijuina T.,Takenawa, T. Application of phosphoinositide-binding domains for the detection and quantification of specific phosphoinositides. Analytical Biochemistry, (2006), 355, 1, 8-18.

Abstract: In mammals, seven phosphoinositides are known to play crucial roles as signaling molecules in a variety of cellular processes. Their synthesis and degradation are thought to be strictly controlled by metabolic enzymes such as phosphoinositide kinases and phosphatases, and their aberrant activities cause diseases. Thus, there is great interest in convenient and high-throughput measurement of such activities for the screening of drugs that enhance or block them. To date, radioactive labeling and colorimetric detection of released inorganic phosphates are mainly used to measure phosphoinositide kinase and phosphatase activities, respectively. Here, we describe a novel method for detecting and quantifying individual phosphoinositides via phosphoinositide-binding domains that exhibit high specificity and affinity toward this lipid. Enzyme-linked immunosorbent assay wells are modified with alkyl chains (C16), which enables more uniform and quantitative immobilization of phosphoinositide-containing liposomes onto the well surfaces. Phosphoinositides, as the substrate or the product, are detected by pleckstrin homology domains that specifically bind to each phosphoinositide. By this method, phosphoinositide contents are measured with higher sensitivities than those by conventional methods. More importantly, both phosphoinositide kinase and phosphatase activities can be measured for purified enzymes and crude cellular lysates. This assay is easy, sensitive, and quantitative and thus may have a variety of applications in the development of diagnostic tests or the screening of therapeutic treatments for diseases such as cancer and diabetes which may be caused by abnormal phosphoinositide metabolism.

Lord M. S., Stenzel, M.H., Simmons, A., Milthorpe, B. K. Lysozyme interaction with poly(HEMA)-based hydrogel. Biomaterials, (2006), 27(8), 1341-1345.

Keywords: Hydrogels; Protein adsorption; Lysozyme; Contact lens

Abstract: Lysozyme interaction with an acrylic-based hydrogel, poly(2-hydroxyethyl methacrylate) co-methacrylic acid (P(HEMA-MAA)), was investigated using a combination of quartz crystal microbalance with dissipation (QCM-D), surface plasmon resonance (SPR) and dual polarisation interferometry (DPI). This combination of techniques demonstrated that lysozyme initially absorbed into the hydrogel matrix and displaced water from the hydrogel while subsequent lysozyme additions were adsorbed onto the surface of the hydrogel material. QCM-D, being sensitive to bound water, showed an overall decrease in mass and stiffening of the layer after lysozyme addition. SPR, a water insensitive technique, showed a net mass increase after addition of lysozyme and buffer rinses. DPI showed that the first exposure of lysozyme to P(HEMA-MAA) was consistent with lysozyme absorption while subsequent lysozyme exposures were consistent with lysozyme adsorption.

Yates, E.,Terry,C., Rees,C., Rudd, T., Duchesne,T., Skidmore, M., Levy, R., Thanh, N., Nichols,R., Clarke, D., and Fernig, D. Protein-GAG Interactions: New Surface-based Techniques, Spectroscopies and Nanotechnology Probes. Biochem. Soc. Trans., (2006), 34 427-430.

Keywords: dual polarization interferometry, fibroblast growth factor, nanoparticle, protein– glycosaminoglycan interaction, quartz crystal microbalance-dissipation, synchrotron radiation CD spectroscopy.

Abstract: New approaches, rooted in the physical sciences, have been developed to gain a more fundamental understanding of protein–GAG (glycosaminoglycan) interactions. DPI (dual polarization interferometry) is an optical technique, which measures real-time changes in the mass of molecules bound at a surface and the geometry of the bound molecules. QCM-D (quartz crystal microbalance-dissipation), an acoustic technique, measures the mass and the viscoelastic properties of adsorbates. The FTIR (Fourier-transform IR) amide bands I, II and III, resulting from the peptide bond, provide insight into protein secondary structure. Synchrotron radiation CD goes to much shorter wavelengths than laboratory CD, allowing access to chromophores that provide insights into the conformation of the GAG chain and of β-strand structures of proteins. To tackle the diversity of GAG structure, we are developing noble metal nanoparticle probes, which can be detected at the level of single particles and so enable single molecule biochemistry and analytical chemistry. These new approaches are enabling new insights into structure–function relationships in GAGs and together they will resolve many of the outstanding problems in this field.

Ricard-Blum, S., Peel, L., Ruggerio, F., Freeman, N. Dual Polarization Interferometry Characterization of Carbohydrate-Protein Interactions. Anal. Biochem., (2006), 352 252-259.

Keywords: Collagen V; Carbohydrate–protein interaction; Heparin binding; Protein–carbohydrate interaction; Dual polarization interferometry, Quantitative analysis

Abstract: Dual polarization interferometry (DPI) is an analytical technique that allows the simultaneous determination of thickness, density, and mass of a biological layer on a sensing waveguide surface in real time. The technique was applied to the analysis of carbohydrate– protein interactions. The selected system involved a 12-kDa recombinant fragment of collagen V (HepV) and heparin, a complex polysaccharide. Here we report on the analysis of thickness, density, and mass of surface structures obtained during the binding of HepV to heparin, which is a useful model compound for the sulfated, protein-binding regions of heparan sulfate. This system, which was initially studied for its biological relevance, displayed anomalous behavior in kinetic studies using surface plasmon resonance (SPR) assays that has been attributed to putative conformational changes. It was this putative conformational change that prompted us to investigate the binding using an alternative analytical approach. While using DPI to monitor binding events, a streptavidin layer (surface coverage 2.105 ng mm ^-2 ) was bound to the sensor surface (92% coverage), which captured 0.105 ng mm ^-2 of biotinylated heparin (a stoichiometric ratio of 1:6 heparin–streptavidin). The heparin inserted into the streptavidin layer but was still found to be capable of binding 0.154 ng mm ^-2 of HepV, which was also observed to insert into the streptavidin layer. This allowed the reliable calculation of the stoichiometric ratio for the HepV–heparin complex (_1.7:1.0), which has proved to be difficult to evaluate by SPR assays. Furthermore, real-time analysis of the heparin–HepV interaction by DPI suggested that there was some surface loss (probably of streptavidin) while the binding was occurring rather than the putative conformational change that has been suggested on the basis of kinetic data alone. This gives further insight into the binding mechanism of HepV to heparin.

Lillis, B., Manning, M., Berney, H., Hurley, E., Mathewson, A., Sheehan, M. Dual Polarisation Interferometry Characterisation of DNA Immobilisation and Hybridisation on a Silanised Support. Biosensors & Bioelectronics, (2006), 21 1459-1467.

Keywords: Dual polarisation; Immobilisation; Hybridisation

Abstract: Dual polarisation interferometry is an analytical technique that allows the simultaneous determination of thickness, density and mass of a biological layer on a sensing waveguide surface in real time. We evaluated, for the first time, the ability of this technique to characterize the covalent immobilisation of single stranded probe DNA and the selective detection of target DNA hybridisation on a silanised support. Two immobilisation strategies have been evaluated: direct attachment of the probe molecule and a more complex chemistry employing a 1,2 homobifunctional crosslinker molecule. With this technique we demonstrate it was possible to determine probe orientation and measure probe coverage at different stages of the immobilisation process in real time and in a single experiment. In addition, by measuring simultaneously changes in thickness and density of the probe layer upon hybridisation of target DNA, it was possible to directly elucidate the impact that probe mobility had on hybridisation efficiency. Direct covalent attachment of an amine modified 19mer resulted in a thickness change of 0.68 nm that was consistent with multipoint attachment of the probe molecule to the surface. Blocking with BSA formed a dense layer of protein molecules that absorbed between the probe molecules on the surface. The observed hybridisation efficiency to target DNA was ∼35%. No further significant reorientation of the probe molecule occurred upon hybridisation. The initial thickness of the probe layer upon attachment to the crosslinker molecule was 0.5 nm. Significant reorientation of the probe molecule surface normal occurred upon hybridisation to target DNA. This indicated that the probe molecule had greater mobility to hybridise to target DNA. The observed hybridisation efficiency for target DNA was ∼85%. The results show that a probe molecule attached to the surface via a crosslinker group is better able to hybridise to target DNA due to its greater mobility.

Lin, S., Lee, C-K., Lin, Y-N.,Lee, S-Y., Sheu, B-C., Tsai J-C., and Hsu, S-M. Homopolyvalent Antibody-Antigen Interaction Kinetic Studies with the Use of a Dual Polarisation Interferometric Biosensor. Biosensors & Bioelectronics, (2006), 22(5) 715-72.

Keywords: Dual-polarization interferometry; C-reactive protein; Binding constant; Affinity; Stoichiometry; Homopolyvalence

Abstract: We used dual-polarization interferometry (DPI) to study the interaction kinetics between a ‘homopolyvalent’ antigen (Ag) and a monoclonal antibody (Ab). A model system, which uses a monoclonal Ab against a homopentameric Ag, C-reactive protein (CRP), is presented with principle and experiments for the study of the interactions between an Ab and an Ag that has multiple identical epitopes. This allows evaluation of the dissociation constant (KD) and of the binding stoichiometry by DPI based on measurements of phase changes of Ab–Ag complexes in the transverse magnetic (TM) and transverse electric (TE) polarization modes. The average experimental value of KD found by the DPI technique for anti-CRP Ab was shown to be in close agreement with the value obtained by an indirect competition-enzyme-linked immunosorbent assay (ELISA). Moreover, the total number of Ab combining sites on the DPI sensor chip was calculated, and the binding stoichiometry of the surface Ag–Ab complex was obtained. This study illustrates the advantages of the DPI method in biosensing in its capacity for simultaneous evaluation of the thickness and refractive index (density, mass) of adsorbed layers. This allowed a comprehensive analysis of affinity reactions between an Ab having two binding sites and a multi-sited Ag.

Lin, S., Lee,C-K., Wang, Y-M., Huang, L-S., Lin, Y-H., Lee S-Y., Sheu, B-C.,Hsu, S. Measurement of dimensions of pentagonal doughnut-shaped C-reactive protein using an atomic force microscope and a dual polarisation interferometric biosensor. Biosensors & Bioelectronics, (2006), 22(2) 323-7.

Keywords: Dual polarization interferometry; Atomic force microscopy; C-reactive protein

Abstract: In order to develop the C-reactive protein (CRP) sensor chips for clinical detection of atherosclerosis and coronary heart disease, we used an atomic force microscope (AFM) and a dual polarization interferometric (DPI) biosensor to probe the surface ultrastructure and to measure the dimensions of CRP. A single pentagonal structure was directly visualized by AFM, and quantitative measurements of the dimensions of the protein were provided. The average height calculated for each pentagonal CRP particle was approximately 3.03±0.37 nm, which basically corresponds to that (36 °A in proto mer diameter) previously obtained from the structure of CRP determined by X-ray crystallography. Moreover, a experiment using dual polarization interferometric (DPI) as a biosensor was then performed, and the average monolayer thickness value (3.18±0.43 nm) that was calculated basically corresponds to that obtained from the experimental value (3.03±0.37 nm) of the height measured by an AFM method for CRP. Further investigations will be performed to study the surface ultrastructure of a single pentagonal CRP molecule, and for this purpose a CRP sample (at low concentration) was scanned in vacuum by AFM. The higher-resolution images clearly revealed the presence of doughnut-shaped CRP molecules. In addition, phase images of CRP molecules were captured simultaneously with their height images, and the lateral dimensions of the doughnut-shaped CRP molecules were then measured. It was found that the average values calculated for the outer diameter (11.13±1.47 nm) and pore diameter (3.52±0.42 nm) are respectively close to those (102 °A in outer diameter and 30 °A in pore d iameter) previously obtained from the structure of CRP determined by X-ray crystallography. This study represents the first direct characterization of the surface ultrastructure and dimensional measurement of the CRP molecule on the sensor chip.

Terry, C. J., Popplewell, J. F., Swann, M. J., Freeman N. J., and Fernig D.G. Characterisation of Membrane mimetics on a dual polarisation interferometer. Biosensors & Bioelectronics, (2006), 22(5) 627-32.

Keywords: Dual polarisation interferometry; Hybrid bilayer membranes

Abstract: Dual polarisation interferometry (DPI) has been used to characterise the formation of hybrid bilayer membranes (HBM) on a silicon-oxynitride surface. This technique allows the simultaneous determination of multiple physical properties of an HBM, as the HBM is being formed in a single experiment: mass, thickness in the z-direction (normal to the surface), tilt angle of the first layer and refractive index. Decanoic acid was covalently attached to an amine modified silicon-oxynitride sensor chip surface via 1-ethyl-3-(3dimethylaminopropyl) carbodiimide hydrochloride condensation reaction. The decanoic acid layer was 0.92±0.12 nm thick, indicating a tilt angle of 57◦ from surface normal, and possessed a mass of 1.05±0.10 ngmm−2 and a refractive index (RI) of 1.450±0.020. Phospholipid vesicles made from 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) were injected over the fatty acid surface to form an HBM. The DPPC HBM was 4.32±0.68 nm thick, with a total mass of 3.18±0.60 ngmm−2 and a RI of 1.404±0.007. The DMPC HBM was 2.12±0.34 nm thick, with a total mass of 2.25±0.51 ngmm−2, and a RI of 1.435±0.007. DPI thus provides an insight into HBM formation and differences between the structural organisation of HBMs of different composition.

Poptoshev, E., and Claessen, P. Adsorption of dimethyldodecylamine-N-oxide at the mica-solution interface studied by ellipsometry Colloids and Surfaces A. Physicochem Eng. Aspects, (2006), 291(1-3) 45-50.

Keywords: Dimethyldodecylamine-N-oxide; Ellipsometry; Mica; Adsorption; Surface excess; Dual polarization interferometry

Abstract: The surface excess of dimethyldodecylamine-N-oxide (DDAO) surfactant at the mica–water interface has been determined using phase modulated ellipsometry. Surface excess versus bulk surfactant concentration isotherms were constructed at different pH values. The plateau adsorbed amount reaches a maximum in the pH range 6–7.5, which includes the native pH of DDAO solutions at the cmc (pH 7.5). At low pH values, where the surfactant is essentially cationic, the surface excess is reduced due to decrease of the mica surface charge, and due to that electrostatic repulsion between charged head groups prevents formation of tightly packed surfactant bilayers. Adsorption from solutions at pH 9 also renders a lower plateau surface excess than that obtained at neutral pH. In this case the surfactant–substrate affinity is greatly reduced due to complete deprotonation of the head group of the surfactant in bulk solution. The results suggest that the degree of protonation of DDAO in the adsorbed layer differs from its bulk value. Close to the negatively charged mica surface the protonation of the head group is increased in order to adjust its charge to that of the surface, whereas the uncharged form is expected to be enriched in the outer part of the bilayer.

Lord, M.S. Biomolecular and Cellular Interactions with Surfaces. PhD Thesis, University of New South Wales, 2006.

Thibault, G., Yudin, J., Wong, P., Tsitrin, V., Sprangers, R., Zhao R., and Houry, W. A., Specificity in substrate and cofactor recognition by the N-terminal domain of the chaperone ClpX. Proc Natl Acad Sci U SA.,(2006), 103(47) 17724-9.

Keywords: NMR _ SspB _ zinc-binding domain

Abstract: Clp ATPases are a unique group of ATP-dependent chaperones supporting targeted protein unfolding and degradation in concert with their respective proteases. ClpX is a representative member of these ATPases; it consists of two domains, a zinc-binding domain (ZBD) that forms dimers and a AAA+ ATP-binding domain that arranges into a hexamer. Analysis of the binding preferences of these two domains in ClpX revealed that both domains preferentially bind to hydrophobic residues but have different sequence preferences, with the AAA+ domain preferentially recognizing a wider range of specific sequences than ZBD. As part of this analysis, the binding site of the ClpX dimeric cofactor, SspB2, on ZBD in ClpX was determined by NMR and mutational analysis. The SspB C terminus was found to interact with a hydrophobic patch on the surface of ZBD. The affinity of SspB2 toward ZBD2 and the geometry of the SspB2–ZBD2 complex were investigated by using the newly developed quantitative optical biosensor method of dual polarization interferometry. The data suggest a model for the interaction between SspB2 and the ClpX hexamer.

Harish, A. Nanomechanical cantilever systems for biological sensing. PhD Thesis, University of Newcastle upon Tyne, 2006.

Suetsugu, S., Kurisu, S.,Oikawa, T.,Yamazaki, D., Oda A., and Takenawa, T., Optimization of WAVE2 complex–induced actin polymerization by membrane-bound IRSp53, PIP3, and Rac. Journal of Cell Biology, (2006), 173(4), 571-585.

Abstract: WAVE2 activates the actin-related protein (Arp) 2/3 complex for Rac-induced actin polymerization during lamellipodium formation and exists as a large WAVE2 protein complex with Sra1/ PIR121, Nap1, Abi1, and HSPC300. IRSp53 binds to both Rac and Cdc42 and is proposed to link Rac to WAVE2. We found that the knockdown of IRSp53 by RNA interference decreased lamellipodium formation without a decrease in the amount of WAVE2 complex. Localization of WAVE2 at the cell periphery was retained in IRSp53 knockdown cells. Moreover, activated Cdc42 but not Rac weakened the association between WAVE2 and IRSp53. When we measured Arp2/3 activation in vitro, the WAVE2 complex isolated from the membrane fraction of cells was fully active in an IRSp53-dependent manner but WAVE2 isolated from the cytosol was not. Purified WAVE2 and purified WAVE2 complex were activated by IRSp53 in a Rac-dependent manner with PIP3-containing liposomes. Therefore, IRSp53 optimizes the activity of the WAVE2 complex in the presence of activated Rac and PIP3.

Tsujita, K., Suetsugu, S., Sasaki,N., Furutani, M.,Oikawa, T., and Takenawa, T. Coordination between the actin cytoskeleton and membrane deformation by a novel membrane tubulation domain of PCH proteins is involved in endocytosis. The Journal of Cell Biology, (2006), 172(2), 269-279.

Abstract: The conserved FER-CIP4 homology (FCH) domain is found in the pombe Cdc15 homology (PCH) protein family members, including formin-binding protein 17 (FBP17). However, the amino acid sequence homology extends beyond the FCH domain. We have termed this region the extended FC (EFC) domain. We found that FBP17 coordinated membrane deformation with actin cytoskeleton reorganization during endocytosis. The EFC domains of FBP17, CIP4, and other PCH protein family members show weak homology to the Binamphiphysin-Rvs (BAR) domain. The EFC domains bound strongly to phosphatidylserine and phosphatidylinositol 4,5-bisphosphate and deformed the plasma membrane and liposomes into narrow tubules. Most PCH proteins possess an SH3 domain that is known to bind to dynamin and that recruited and activated neural Wiskott-Aldrich syndrome protein (N-WASP) at the plasma membrane. FBP17 and/or CIP4 contributed to the formation of the protein complex, including N-WASP and dynamin-2, in the early stage of endocytosis. Furthermore, knockdown of endogenous FBP17 and CIP4 impaired endocytosis. Our data indicate that PCH protein family members couple membrane deformation to actin cytoskeleton reorganization in various cellular processes.

Halthur,T., Claessen, P., Elofsson, U. Immobilization of Enamel Matrix Derivate Protein onto Polypeptide Multilayers. Comparative in Situ Measurements Using Ellipsometry, Quartz Crystal Microbalance with Dissipation, and Dual-Polarization Interferometry. Langmuir, (2006), 22(26)11065-71.

Abstract: The buildup of biodegradable poly(L-glutamic acid) (PGA) and poly(L-lysine) (PLL) multilayers on silica and titanium surfaces and the immobilization of enamel matrix derivate (EMD) protein was followed by utilizing in situ ellipsometry, quartz crystal microbalance with dissipation, and dual-polarization interferometry (DPI). The use of the relatively new DPI technique validated earlier published ellipsometry measurements of the PLL-PGA polypeptide films. The hydrophobic aggregating EMD protein was successfully immobilized both on top of and within the multilayer structures at pH 5.0. DPI measurements further indicated that the immobilization of EMD is influenced by the flow pattern during adsorption. The formed polypeptide-EMD multilayer films are of interest since it is known that EMD is able to trigger cell response and induce biomineralization. The multilayer films thus have potential to be useful as bioactive and biodegradable coatings for future dental implants.

Freeman, N., Dual Polarisation Interferometry: An Optical Technique to Measure the Orientation and Structure of Proteins at the Solid-Liquid Interface in Real Time. Proteins at Solid-Liquid Interfaces. Ed. Philippe Déjardin, (2006).

Chen, X., Pelton,R. Pre-Adsorption of Amphiphilic Polymers on Synthetic Surfaces for Biofouling Retardation, Adv. Materials Research, (2006), 11(12) 363-366.

Keywords: Surface treatment, Amphiphilic polymer, Protein adsorption

Abstract. Polystyrene (PS), polyethylene (PE), polypropylene (PP), glass and stainless steel were exposed to aqueous solutions of a series amphiphilic polymers at room temperature, including N-isopropylacrylamide (NIPAM)-based polymers, polyvinylpyrrolidone (PVP), polypropylene oxide (PPO)-polyethylene oxide (PEO) block copolymers and PEO. Dynamic contact angle measurements of the material surfaces before and after the treatment indicate that only NIPAM-based polymers can adsorb on both hydrophobic and hydrophilic surfaces. The surface morphologies of the materials before and after polymer adsorption were investigated by profilometry. Protein adsorption on the surfaces pre-adsorbed NIPAM-based polymers was investigated by dual polarisation interferometry (DPI) and profilometry using lysozyme as the model protein. The results obtained indicate that NIPAM-based polymers can significantly improve the biofouling resistance of synthetic surfaces.

Suetsugu, S., Murayama, K., Sakamoto, A., Hanawa-Suetsugu, K., Seto, A., Oikawa, T., Mishima, C., Shirouzu, Tadaomi , M., Yokoyama, S. The RAC Binding Domain/IRSp53-MIM Homology Domain of IRSp53 Induces RAC-dependent Membrane Deformation. J. Biol. Chem., (2006), 281(46), 35347-35358.

Abstract: The concave surface of the crescent-shaped Bin-amphiphysin-Rvs (BAR) domain is postulated to bind to the cell membrane to induce membrane deformation of a specific curvature. The Rac binding (RCB) domain/IRSp53-MIM homology domain (IMD) has a dimeric structure that is similar to the structure of the BAR domain; however, the RCB domain/IMD has a “zeppelin-shaped” dimer. Interestingly, the RCB domain/IMD of IRSp53 possesses Rac binding, membrane binding, and actin filament binding abilities. Here we report that the RCB domain/IMD of IRSp53 induces membrane deformation independent of the actin filaments in a Racdependent manner. In contrast to the BAR domain, the RCB domain/IMD did not cause long tubulation of the artificial liposomes; however, the Rac binding domain caused the formation of small buds on the liposomal surface. When expressed in cells, the Rac binding domain induced outward protrusion of the plasma membrane in a direction opposite to that induced by the BAR domain. Mapping of the amino acids responsible for membrane deformation suggests that the convex surface of the Rac binding domain binds to the membrane in a Rac-dependent manner, which may explain the mechanism of the membrane deformation induced by the RCB domain/IMD.

Yuji O, K., Yoji, S., Tomoharu, M., Kota K., Kazuo K. Metal Ion binding to Prion Protein. Biophysics, (2006), 46(2), S439.

Abstract: Prion diseases are fatal neurodegenerative diseases caused by conformational transitionof the cellular isoform of prion protein (PrPC) to the pathogenic scrapie isoform PrPSc). Function of the PrPC is still not clear. But, it is known that the prion protein is a copper binding protein and it is suggested that PrPC is involved in the copper metabolism. We have applied Dual Polarisation Interferometry (DPI) toto study the interaction between the recombinant mouse prion PrPCand nine different divalent metal ions. Mass, thickness and refractive index changes induced by the metal ion binding were observed. We have estimated thebinding constants, association and dissociation rate constants, and molecular ratios of protein to metal ions. We found that copper has the highest affinity of PrPC, but some other metal ions also have a certain degrees of affinity to this protein. This result suggests that PrPC may participate in the metabolism of not only copper but also some other divalent metal ions.

Yoji , Y., Toshifusa ,T. Studies of metal ion interactions with calmodulin using Dual Polarization Interferometry (DPI). Biophysics,(2006),46(2), S271.

Abstract: A number of optical biosensors have been developed in rapid succession for the past decade. They have been adopted as a tool for drug discovery and interaction analysis in a wide area of biological researches. In the present study, calmodulin (CaM) interactions with metal ions were investigated using the dual polarisation interferometry (DPI), a novel technology of optical biosensor to measure thickness, refractive index and mass on a solid-liquid interface. CaM, a key protein in intracellular calcium signalling, was immobilised on the sensor surface in a ca. 1.4 nm layer. Subsequently, the CaM layer was subjected to interaction by exposing to micro-flow of metal ions at several concentrations. The metal ions used in this study were Na ²⁺ , Mg ²⁺ , Al ³⁺ , Ca ²⁺ , Mn ²⁺ , Fe ²⁺ , Co ²⁺ , Ni ²⁺ , Cu ²⁺ , Zn ²⁺ , Cd ²⁺ , Sn ²⁺ and Hg ²⁺ . In addition to Ca ²⁺ , as a result, Co ²⁺ , Ni ²⁺ , Zn ²⁺ , Cu ²⁺ and Cd ²⁺ showed strong interactions with CaM. When CaM interacted with Co ²⁺ , Ni ²⁺ , Zn ²⁺ , Cu ²⁺ and Cd ²⁺ , the thickness of the CaM layer was decreased and the refractive indices were increased, whereas Ca ²⁺ interaction showed increase in thickness and decrease in refractive index. This suggests that the conformational changes to CaM by Ca ²⁺ are different from those by other ions. The dissociation constant, association rate constant and dissociation rate constant of CaM with Ca ²⁺ were estimated to be 26 microM, 3.5*103 M ^-1 s ^-1 and 0.091s ^-1 , respectively. The present study proved the DPI technique to provide significant information about protein structures on metal ion interaction.

Lee, T. H., Aguilar, M.I. Trends in the development and application of functional biomembrane surfaces. Biotechnology Annual Review, (2006), Volume 12, Ed. M. Rafaat El-Gewely pp 85-136.

Keywords: bilayer lipid membranes; supported lipid monolayer; tethered lipid bilayers; immobilised phospholipid monolayer; gel-entrapped liposomes; membrane microarray; nanoarray.