Ordered spherical cap gold cavity arrays with 5.4, 1.6, and 0.98 μm diameter apertures were explored as capture surfaces for human blood platelets to investigate the impact of surface geometry and chemical modification on platelet capture efficiency and their potential as platforms for surface enhanced Raman spectroscopy of single platelets. The substrates were chemically modified with single-constituent self-assembled monolayers (SAM) or mixed SAMs comprised of thiol-functionalized arginine–glycine–aspartic acid (RGD, a platelet integrin target) with or without 1-octanethiol (adhesion inhibitor). As expected, platelet adhesion was promoted and inhibited at RGD and alkanethiol modified surfaces, respectively. Platelet adhesion was reversible, and binding efficiency at the peptide modified substrates correlated inversely with pore diameter. Captured platelets underwent morphological change on capture, the extent of which depended on the topology of the underlying substrate. Regioselective capture of the platelets enabled study for the first time of the surface enhanced Raman spectroscopy of single blood platelets, yielding high quality Raman spectroscopy of individual platelets at 1.6 μm diameter pore arrays. Given the medical importance of blood platelets across a range of diseases from cancer to psychiatric illness, such approaches to platelet capture may provide a useful route to Raman spectroscopy for platelet related diagnostics.Keywords: blood platelet; gold microcavity array; integrin αIIbβ3; RGD; surface; surface-enhanced Raman spectroscopy; thrombin;

A novel and versatile approach to electrichemically triggering the release of a reagent, β-cyclodextrin (β-CD), selectively to the proximal leaflet of a supported lipid bilayer is described. Selective delivery is achieved by creating a spanning lipid bilayer across a microcavity array and exploiting the irreversible redox disassembly of the host–guest complex formed between thiolated ferrocene (Fc) and β-cyclodextrin (β-CD) in the presence of chloride. Self-assembled monolayers of the ferrocene–alkanethiols were formed regioselectively on the interior surface of highly ordered 2.8 μm cavities while the exterior top surface of the array was blocked with a monolayer of mercaptoethanol. The Fc monolayers were complexed with β-CD or β-CD-conjugated to streptavidin (β-CD-SA). Phospholipid bilayers were then assembled across the array via combined Langmuir–Blodgett/vesicle fusion leading to a spanning bilayer suspended across the aqueous filled microcavities. Upon application of a positive potential, ferrocene is oxidized to ferrocinium cation, disrupting the inclusion complex and leading to the release of the β-CD into the microcavity solution where it diffuses to the lower leaflet of the suspended bilayer. Disassembly of the supramolecular complex within the cavities and binding of the β-CD-SA to a biotinylated bilayer was followed by voltammetry and impedance spectroscopy where it caused a large increase in membrane resistance. For unmodified β-CD, the extraction of cholesterol from a cholesterol containing bilayer was evident in a decrease in the bilayer resistance. For the first time, this direct approach to targeted delivery of a reagent to the proximal layer of a lipid bilayer offers the potential to build models of bidirectional signaling (inside-out vs outside-in) in cell membrane model systems.

Deeper understanding of the molecular interactions between polymeric materials and the lipid membrane is important across a range of applications from permeation for drug delivery to encapsulation for immuno-evasion. Using highly fluidic microcavity supported lipid bilayers, we studied the interactions between amphiphilic polymer PP50 and a DOPC lipid bilayer. As the PP50 polymer is pH responsive the studies were carried out at pH 6.5, 7.05 and 7.5, corresponding to fully, partly protonated (pH = pKa = 7.05) and fully ionized states of the polymer, respectively. Fluorescence correlation spectroscopy (FCS) using both labelled lipid and polymer revealed the PP50 associates with the bilayer interface across all pHs where its diffusion along the interface is impeded. Both FCS and electrochemical impedance spectroscopy (EIS) data indicate that the PP50 does not penetrate fully into the bilayer core but rather forms a layer at the bilayer aqueous interface reflected in increased resistance and decreased capacitance of the bilayer on PP50 binding. The extent of these effects and the dynamics of binding are influenced by pH, increasing with decreasing pH. These experimental trends concurred with coarse grained Monte Carlo simulations of polymer–bilayer interactions wherein a model hydrophilic polymer backbone grafted with side chains of varying hydrophobicity, to mimic the effect of varying pH, was simulated based on the bond fluctuation model with explicit solvent. Simulation results showed that with increasing hydrophobicity, the polymer penetrated deeper into the contacting bilayer leaflet of the membrane suppressing, consistent with EIS data, solvent permeation and that a full insertion of the polymer into the bilayer core is not necessary for suppression of permeability.

The synthesis and photophysical properties of water responsive 1,10-phenanthrolyl and 2,2′-bipyridyl substituted BODIPY derivatives prepared as lipid probes for cell imaging are reported. These compounds exhibit intense emission in non-aqueous media that is reversibly extinguished in aqueous media. Halogen substitution at the BODIPY indacene core decreases the emission quantum yields and causes red spectral shifts of emission maxima of the order H > Br > I. The emission was quenched on binding of the phenanthrolyl or bipyridyl to cations Fe2+, Cu2+ and Zn2+. The origin of the water switching effect and the impact of halogen substitution was investigated by modelling the electronic structure of the fluorophore using DFT methods. All compounds showed excellent permeability to live cells and were found, under imaging conditions, to generally exhibit low cytotoxicity. The absence of emission in the aqueous environment facilitated the collection of high contrast images from membranous regions and lipid droplets in live cells. The staining pattern in HeLa cells was found to depend on halogen substitution. Across both bpy and phen derivatives the halogenated probes showed the strongest targeting of lipid droplets within cells whilst the parent unsubstituted compounds were more widely dispersed in the cytoplasm. Resonance Raman imaging was used to map the distribution of probes within the cell and confirmed that the compounds showed strong co-localisation with lipid rich regions of the cell.

A new approach for the fabrication of luminescent ratiometric sensing nanosensors is described using core–shell nanoparticles in which the probe and reference are spatially separated into the shell and core of the nanostructure respectively. The isolation of the reference in the core of the particle ensures a stable emission reference signal unaffected by the external environment. The core shell structure was prepared by engineering structurally well-defined Ru-conjugated block copolymers which acted as emulsifiers in the miniemulsion polymerisation of BODIPY loaded styrene nanoparticles. The resulting particles are highly stable and show excellent size monodispersity. The nanosensors exhibit dual emission under a single excitation wavelength with a reversible and quantitative ratiometric response to the O2 content in aqueous media. In the presence of a low concentration of CTAB, the particles cross the cell membrane and the particles show negligible cytotoxicity. Such an approach to sensor nanoparticles should be of value across a range of applications where a stable ratiometric signal in diverse environments is required.

•Polyoxometalates have attractive photochemical and redox properties and increasingly diverse structures are being developed.•Their oxidative photocatalysis reactions can sensitized for visible excitation in charge transfer materials.•Overview of photophysics of polyoxometalate in charge-transfer salts.•Review of recent advances in hybrid polyoxometalate photochemical assemblies.•Applications, and future perspectives.Polyoxometalate (POM) based supramolecular assemblies have received significant attention over recent years because of their unique and diverse redox and photochemical behaviour and their potential value across a range of important light driven applications such as photo-driven synthesis and photocatalysis. This review explores the dominant approaches to assembly of polyoxometalates into supramolecular materials, both covalent and electrostatic, with particular focus on charge transfer materials and those capable of sensitized photoelectrocatalysis. The integration of POMs as components in devices such as dye sensitized solar cells, electrocatalytic photoanodes, sensing and waste remediation are considered.

A highly efficient and versatile route to the preparation of tris(heteroleptic) Ru(II) polypyridyl complexes is described which permits access to two or more independently conjugatable termini in the final structure. The strategy utilizes the well-known Ru(DMSO)4Cl2 precursor to form the Ru(N^N)(DMSO)2Cl2 product and then proceeds through an oxalate intermediate which can be cleaved under acidic conditions to control the stoichiometric addition of polypyridyl ligands to the Ru(II) coordination sphere enabling the stepwise assembly of the heteroleptic complex. To exemplify this approach, three complexes were prepared including the novel: [Ru(dppz)(bpyArCOOH)(bpyArCOOEt)]2+ (where dppz is dipyridophenazine, bpyArCOOH and bpyArCOOEt are 4-(4-carboxyphenyl)- and 4-(4-ethoxycarbonylphenyl)-2,2-bipyridine, respectively) in which the synthetic yield from the RuCl3 starting material to final product is 82%. A sequential conjugation–deprotection–conjugation step is then described to yield a Ru(II) complex which is both PEGylated and peptide-conjugated. This synthetic approach offers a useful means to expand the structural diversity of bis coordinated Ru(II) polypyridyl complexes and provides a simple route to building multifunctionality into such complexes which should broaden their applications, in particular in the domain of bioimaging and therapy.

•Gold cavity arrays support stable, fluidic and solvent free lipid bilayers in presence of ion gradient.•The pore dimensions make them suited to both electrochemical and microscopy interrogation.•EIS sensitively reports membrane resistance and capacitance changes on Nigericin or Valinomycin reconstitution.Microcavity supported lipid bilayers, MSLBs, were applied to an electrochemical investigation of ionophore mediated ion transport. The arrays comprise of a 1 cm2 gold electrode imprinted with an ordered array of uniform spherical-cap pores of 2.8 μm diameter prepared by gold electrodeposition through polystyrene templating spheres. The pores were pre-filled with aqueous buffer prior to Langmuir-Blodgett assembly of a 1,2-dioleoyl-sn-glycero-3-phosphocholine bilayer. Fluorescence lifetime correlation spectroscopy enabled by the micron dimensions of the pores permitted study of lipid diffusion across single apertures, yielding a diffusion coefficient of 12.58 ± 1.28 μm2 s− 1 and anomalous exponent of 1.03 ± 0.02, consistent with Brownian motion. From FLCS, the MSLBs were stable over 3 days and electrochemical impedance spectroscopy of the membrane with and without ionic gradient over experimental windows of 6 h showed excellent stability. Two ionophores were studied at the MSLBs; Valinomycin, a K+ uniporter and Nigericin, a K+/H+ antiporter. Ionophore reconstituted into the DOPC bilayer resulted in a decrease and increase in membrane resistance and capacitance respectively. Significant increases in Valinomycin and Nigericin activity were observed, reflected in large decreases in membrane resistance when K+ was present in the contacting buffer and in the presence of H+ ionic gradient across the membrane respectively.

A ruthenium(II) polypyridyl–BODIPY dyad is presented which exhibits a solvent switchable dual emission. Intense oxygen sensitive emission from the ruthenium centre and O2 independent emission from the BODIPY centre, are both observed in organic media. In aqueous media, the BODIPY emission is reversibly switched off leaving only a ruthenium centred emission. The materials are interesting both as self-referenced O2 probes and for cell/tissue imaging.

A key prerequisite in an ideal supported lipid bilayer based cell membrane model is that the mobility of both the lipid matrix and its components are unhindered by the underlying support. This is not trivial and with the exception of liposomes, many of even the most advanced approaches, although accomplishing lipid mobility, fail to achieve complete mobility of incorporated membrane proteins. This is addressed in a novel platform comprising lipid bilayers assembled over buffer-filled, arrays of spherical cap microcavities formed from microsphere template polydimethoxysilane. Prior to bilayer assembly the PDMS is rendered hydrophilic by plasma treatment and the lipid bilayer prepared using Langmuir Blodgett assembly followed by liposome/proteoliposome fusion. Fluorescence Lifetime Correlation Spectroscopy confirmed the pore suspended lipid bilayer exhibits diffusion coefficients comparable to free-standing vesicles in solution. The bilayer modified arrays are highly reproducible and stable over days. As the bilayers are suspended over deep aqueous reservoirs, reconstituted membrane proteins experience an aqueous interface at both membrane interfaces and attain full lateral mobility. Their utility as membrane protein platforms was exemplified in two case studies with proteins of different dimensions in their extracellular and cytoplasmic domains reconstituted into DOPC lipid bilayers; Glycophorin A, and Integrin αIIbβ3. In both cases, the proteins exhibited 100% mobility with high lateral diffusion coefficients.

A first investigation into the application of a luminescent osmium(II) bipyridine complex to live cell imaging is presented. Osmium(II) (bis-2,2-bipyridyl)-2(4-carboxylphenyl) imidazo[4,5f][1,10]phenanthroline was prepared and conjugated to octaarginine, a cell penetrating peptide. The photophysics, cell uptake and cytotoxicity of this osmium complex conjugate were performed and compared with its ruthenium analogue. Cell uptake and distribution of both ruthenium and osmium conjugates were very similar with rapid transmembrane transport of the osmium probe (complete within approx. 20 min) and dispersion throughout the cytoplasm and organelles. The near-infrared (NIR) emission of the osmium complex (λmax 726 nm) coincides well with the biological optical window and this facilitated luminescent and luminescence lifetime imaging of the cell which was well resolved from cell autofluorescence. The large Stokes shift of the emission also permitted resonance Raman mapping of the dye within CHO cells. Rather surprisingly, the osmium conjugate exhibited very low cytotoxicity when incubated both in the dark and under visible irradiation. This was attributed to the remarkable stability of this complex which was reflected by the complete absence of photo-bleaching of the complex even under extended continuous irradiation. In addition, when compared to its ruthenium analogue its luminescence was short-lived in water therefore rendering it insensitive to O2.

Platelet integrin αIIbβ3 is a key mediator of platelet activation and thrombosis. Upon activation αIIbβ3 undergoes significant conformational rearrangement, inducing complex bidirectional signalling and protein recruitment leading to platelet activation. Reconstituted lipid models of the integrin can enhance our understanding of the structural and mechanistic details of αIIbβ3 behaviour away from the complexity of the platelet machinery. Here, a novel method of αIIbβ3 insertion into Giant Unilamellar Vesicles (GUVs) is described that allows for effective integrin reconstitution unrestricted by lipid composition. αIIbβ3 was inserted into two GUV lipid compositions that seek to better mimic the platelet membrane. First, “nature's own”, comprising 32% DOPC, 25% DOPE, 20% CH, 15% SM and 8% DOPS, intended to mimic the platelet cell membrane. Fluorescence Lifetime Correlation Spectroscopy (FLCS) reveals that exposure of the integrin to the activators Mn2+ or DTT does not influence the diffusion coefficient of αIIbβ3. Similarly, exposure to αIIbβ3's primary ligand fibrinogen (Fg) alone does not affect αIIbβ3's diffusion coefficient. However, addition of Fg with either activator reduces the integrin diffusion coefficient from 2.52 ± 0.29 to μm2 s−1 to 1.56 ± 0.26 (Mn2+) or 1.49 ± 0.41 μm2 s−1 (DTT) which is consistent with aggregation of activated αIIbβ3 induced by fibrinogen binding. The Multichannel Scaler (MCS) trace shows that the integrin–Fg complex diffuses through the confocal volume in clusters. Using the Saffman–Delbrück model as a first approximation, the diffusion coefficient of the complex suggests at least a 20-fold increase in the radius of membrane bound protein, consistent with integrin clustering. Second, αIIbβ3 was also reconstituted into a “raft forming” GUV with well defined liquid disordered (Ld) and liquid ordered (Lo) phases. Using confocal microscopy and lipid partitioning dyes, αIIbβ3 showed an affinity for the DOPC rich Ld phase of the raft forming GUVs, and was effectively excluded from the cholesterol and sphingomyelin rich Lo phase. Activation and Fg binding of the integrin did not alter the distribution of αIIbβ3 between the lipid phases. This observation suggests partitioning of the activated fibrinogen bound αIIbβ3 into cholesterol rich domains is not responsible for the integrin clustering observed.

A novel mitochondrial localizing ruthenium(II) peptide conjugate capable of monitoring dynamic changes in local O2 concentrations within living cells is presented. The complex is comprised of luminescent dinuclear ruthenium(II) polypyridyl complex bridged across a single mitochondrial penetrating peptide, FrFKFrFK-CONH2 (r = d-arginine). The membrane permeability and selective uptake of the peptide conjugate at the mitochondria of mammalian cells was demonstrated using confocal microscopy. Dye co-localization studies confirmed very precise localization and preconcentration of the probe at the mitochondria. This precision permitted collection of luminescent lifetime images of the probe, without the need for co-localizing dye and permitted semiquantitative determination of oxygen concentration at the mitochondria using calibration curves collected at 37 °C for the peptide conjugate in PBS buffer. Using Antimycin A the ability of the probe to respond dynamically to changing O2 concentrations within live HeLa cells was demonstrated. Furthermore, based on lifetime data it was evident that the probe also responds to elevated reactive oxygen species (ROS) levels within the mitochondria, where the greater quenching capacity of these species led to luminescent lifetimes of the probe at longer Antimycin A incubation times which lay outside of the O2 concentration range. Although both the dinuclear complex and a mononuclear analogue conjugated to an octaarginine peptide sequence exhibited some cytotoxicity over 24 h, cells were tolerant of the probes over periods of 4 to 6 h which facilitated imaging. These metal-peptide conjugated probes offer a valuable opportunity for following dynamic changes to mitochondrial function which should be of use across domains in which the metabolic activity of live cells are of interest from molecular biology and drug discovery.

The ability of two novel ruthenium(II) polypyridyl-Arg-Gly-Asp (RGD) peptide conjugates to act as molecular probes for reporting on the presence and conformation of integrin αIIbβ3 in solution and in live cells was described. The compounds are [Ru(bpy)2PIC-RGD]2+, bpy-RGD, and [Ru(dpp)2PIC-RGD]2+, dpp-RGD, where dpp is 4,7-diphenyl-1,10-phenanthroline, bpy is 2,2′-bipyridine, and PIC is 2-(4-carboxyphenyl)imidazo[4,5-f][1,10]phenanthroline. Bpy-RGD is hydrophilic, whereas dpp-RGD is comparatively hydrophobic. Both probes exhibited good affinity and high specificity for purified αIIbβ3 in solution. Binding of either complex to the resting integrin resulted in an approximately 8-fold increase of emission intensity from the metal center with dissociation constants (Kd) in the micromolar range for each complex. The Kd for each conjugate/αIIbβ3 assembly were compared following treatment of the integrin with the activating agents, Mn2+ and dithiothreitol (DTT), which are commonly used to induce active-like conformational changes in the integrin. For bpy-RGD/αIIbβ3 Kd showed relatively little variation with integrin activation, presenting the following trend: denatured αIIbβ3 > resting αIIbβ3 = pretreated DTT = pretreated Mn2+. Kd for dpp-RGD/ αIIbβ3 showed greater variation with integrin activation and the following trend was followed: denatured αIIbβ3 > resting αIIbβ3 > pretreated Mn2+ = pretreated DTT. Time resolved luminescence anisotropy was carried out to obtain the rotational correlation time of bpy-RGD and dpp-RGD bound to resting or nominally activated integrin. The rotational correlation times of bpy-RGD and dpp-RGD, too fast to measure unbound, decreased to 1.50 ± 0.03 μs and 2.58 ± 0.04 μs, respectively, when the complexes were bound to resting integrin. Addition of Mn2+ to bpy-RGD/αIIbβ3 or dpp-RGD/αIIbβ3 reduced the rotational correlation time of the ruthenium center to 1.29 ± 0.03 μs and to 1.72 ± 0.03 μs, respectively. Following treatment, the rotational correlation time decreased to 1.04 ± 0.01 μs and 1.29 ± 0.03 μs for bpy-RGD/αIIbβ3, and dpp-RGD/αIIbβ3, respectively. The large relative changes in rotational correlation times observed for Mn2+ or DTT activated integrin indicates significant change in protein conformation compared with the resting integrin. The results also indicated that the metal complex itself affects the final conformational and/or aggregation status of the protein obtained. Furthermore, the extent of conformational change was influenced by whether the probe was bound to the integrin before or after activator treatment. Finally, in vitro studies indicated that both probes selectively bind to CHO cells expressing the resting form of αIIbβ3. In each case the probe colocalized with αIIb specific SZ22 antibody. Overall, this work indicates that bpy-RGD and dpp-RGD may be useful peptide-probes for rapid assessment of integrin structural status and localization in solution and cells.

Blood platelet adhesion is crucial in dictating haemocompatibility of medical implants and in platelet capture in diagnostics. Understanding the role of platelet activation in dictating platelet adhesion at chemically modified interfaces is important but relatively unexplored. Using scanning electron microscopy and confocal fluorescence microscopy a quantitative assessment of capture of blood platelets at self-assembled monolayers and mixed monolayers (SAMs) on gold as a function of the activation status of the platelets was conducted. Single and mixed monolayers were prepared using thiol-functionalized arginine-glycine-aspartic acid (RGD), C-Ahx-GRGDS (Ahx = aminohexanoic acid linker), thiolated poly(ethylene)glycol (PEG-COOH) and 1-octanethiol. When incubated with suspensions of resting platelets, RGD promoted platelet adhesion compared to bare or alkanethiol modified gold. Increasing the alkanethiol ratio in the deposition solution decreased the extent of platelet adhesion. Platelet adhesion increased approximately 3 fold at PEG-COO- modified surfaces compared to RGD-alone. Platelets adhered to RGD or mixed RGD:alkane SAM surfaces were found to be captured in their resting state. In contrast, platelets captured at PEG-COO- SAM surfaces were activated by these substrates. The effect of treating platelets with the chemical activators, Mn2+ or DTT or the physiological activator, thrombin, on the capture efficiency and activation at RGD modified surfaces was also investigated. Mn2+ treated platelets presented similar adhesion to untreated platelets, while surprisingly DTT yielded a very significant decrease in platelet adhesion. And, any platelets that were captured, were in a resting state. Thrombin activated platelets were captured with similar efficiencies as untreated platelets. However, the platelets captured were fully activated. The distinction between capture of chemically and physiologically activated platelet is interesting and likely to originate from differences in the conformation of the integrin induced by each process. Finally, platelet adhesion to each surface could be reversed by incubation with a solution of linear or cyclical RGD or PEG-COO- for the RGD and PEGCOO- surfaces respectively. The specificity of platelet removal confirmed that platelet adhesion at RGD surfaces is occurring through integrin–RGD interactions.

Spontaneously adsorbed monolayers have been formed on gold electrodes using a Keggin polyoxotungstate with covalently attached alkanethiol linkers of two different lengths. Films of both polyoxotungstates show two well-defined reduction processes associated with the polyoxotungstate centers where the ionic liquid, [BMIM][BF4], acts as supporting electrolyte. The surface coverages are both less than that expected for a close-packed monolayer. For the short and long linkers, the voltammetric response can be described in terms of the Butler–Volmer response involving a surface confined species using standard heterogeneous electron transfer rate constants of 170 and 140 s–1 for the first reduction and 150 and 100 s–1 for the second reduction processes, respectively. The rate of electron transfer to a solution phase redox probe, ferrocyanide, is significantly more sensitive to the length of the linker than the rate of electron transfer to the tungstate centers. This behavior probably arises due to potential-induced changes in the film structure.

The targeted delivery of luminescent Ru(II) polypyridyl complexes to the nucleus of live mammalian cells by a Nuclear Localisation Signal (NLS) peptide; NF-κB is demonstrated.

The synthesis and characterisation of iridium(III) bis(2-(2,4-difluorophenyl)pyridinato-N, C2′)-2(4-carboxylphenyl)imidazo[4,5-f][1,10]phenanthroline perchlorate, [Ir(dfpp)2(picCOOH)]+ and its octaarginine conjugate [Ir(dfpp)2(picCONH-Arg8)]9 + are reported. Both complex and conjugate exhibit intense and long-lived luminescence, which is O2 and pH sensitive. Conjugation to the polyarginine peptide renders the complex very water soluble. The uptake of the parent iridium(III) complex and conjugate are compared in two mammalian cell lines; SP2 myeloma and Chinese hamster ovary (CHO). Both complexes internalise into the cytoplasm, however dye uptake rate and distribution vary with peptide conjugation and with cell identity. Whereas transmembrane transport is thought to have been facilitated by the dimethyl sulfoxide (DMSO) used as co-solvent (0.05% v/v) for the parent complex, the octaarginine, the dye-conjugate (iridium-R8) is membrane permeable in water only. Both complexes exhibit high cytotoxicity, evident through blebbing and vacuole formation within living cells, indicative of apoptosis, within 30 min of exposure to the probe. The IC50 recorded for the cells in the dark was independent, in the case of the parent complex, of the identity of the cell, with IC50 of 84.8 μM and 88 μM respectively for SP2 and CHO cells. The IC50 approximately doubled for the polyarginine conjugate and displayed a significant dependence on cell type with IC50 of 35 μM and 54.1 μM respectively for SP2 and CHO cells. These IC50 values were recorded in the dark. However under irradiation cell death is considerably faster. Evidence from imaging suggests that the conjugate penetrates the nucleus whereas the parent does not, indicating that nuclear penetration may play a role in cytotoxicity.Conjugation of octaarginine polypeptide to a cyclometalated Ir(III) complex renders it water soluble, cell permeable and effectively doubles the cytotoxicty of the metal complex.Highlights► A novel, highly luminescent, cyclometalated Ir(III) polypyridyl complex and its octaarginine peptide conjugate are reported. ► The octaarginine conjugate is highly water soluble and crosses the cell membrane without the need for permeabilization. ► The peptide conjugated complex rapidly concentrates inside live mammalian cells, but uptake and distribution varies with cell type. ► Cell uptake of the conjugate by CHO is almost double that of the SP2 cell line, but cytotoxcity is higher in the latter. ► The octaarginine tail increases the cytotoxicity of the iridium by almost two-fold by comparison with the parent complex.

•Self-assembled films of [Ru(bpy)3]2γ*-[W18O54(SO4)2] were prepared at Pt electrodes.•Low electrolyte concentrations are required to maintain film integrity.•Photocatalysis by [W18O54(SO4)2]4− is sensitized by the ruthenium centres.•Photocatalytic performance of the film was optimised under low electrolyte conditions.•The highest photocurrent response from POM films under visible irradiation is reported.Under appropriate conditions, photocatalytic oxidation of benzyl alcohol by Dawson polyoxometalates, POM, can be sensitized by ruthenium(II) polypyridyl complexes under visible irradiation of electrostatic adducts formed between these materials. To realize the potential of such sensitization in a photoelectrochemical cell, thin film assembly of at an electrode interface of such materials is required. However, because they are electrostatically associated, the structural integrity of the Ru-POM complex can be compromised by the contacting electrolyte. Herein, thin layers of an electrostatic adduct formed between the polyoxotungstate γ*-[W18O54(SO4)2]4− and the ruthenium polypyridyl complex [Ru(bpy)3]2+ have been self-assembled on platinum electrodes for the first time, using alternate immersion layer-by-layer assembly. SEM and AFM imaging reveal that the adduct films exhibit a highly nanoporous structure with an average pore diameter of approximately 200 nm; a film morphology that is clearly distinctive from films comprised of either parent ion; [(Hex)4N]4γ*-[W18O54(SO4)2] or [Ru(bpy)3](PF6)2 alone, and indicate that ion-pairing is what drives film porosity. Cyclic voltammetry of the adduct ([Ru–POM]) films was performed on Pt microelectrodes in the absence of, or presence of low levels of added supporting electrolyte to minimise any electrostatic disruption of the ionic film. Low concentrations of supporting electrolyte (1 mM (Bu)4NBF4) decreased the resistance across the cell by an order of magnitude, compared with no electrolyte whilst maintaining the film integrity. Photoelectrochemical studies of [Ru–POM] films using visible light (λ > 400 nm) and benzyl alcohol as the sacrificial electron donor were performed in the absence of added electrolyte. Resonance Raman spectroscopy, along with the extinction of photocurrent on oxidation of the ruthenium center indicate that the benzyl alcohol oxidation is sensitized in the visible spectral region by the ruthenium center. Increasing the surface coverage by adding more layers resulted in a net decrease in photocurrent density due to ion-transport limitations. However, rates of electrochemical re-oxidation of the photochemically reduced films ([Ru–POM]red) increased exponentially with increasing applied overpotential up to the oxidation potential of the ruthenium center. The overall photocurrent was optimized at 59 μA cm−2 in (1 mM (Bu)4NBF4), which is almost an order of magnitude higher than currents generated under the same conditions (at 6.8 μA cm−2) but in the absence of added electrolyte.Graphical abstractThe photocatalytic oxidation of benzylalcohol, sensitized under visible irratdiation at a layer by layer assembled film of γ*-[W18O54(SO4)2]4− and [Ru(bpy)3]2+ is optimised in low electrolyte media under which conditions the electrostatically associated film is stable.

A novel Near Infra-Red emitting BODIPY derivative is presented which exhibits the largest Stokes shift thus far reported for a BODIPY compound.

Fluorescence lifetime correlation spectroscopy (FLCS) has been used to probe the influence of PEG-8000 on the fluidity of fluorescently labeled planar supported lipid bilayers on ozone plasma treated glass. The lipid membrane compositions examined were; DOPC, DOPC/DOPS (80/20 mol/mol) and DMPC, with and without cholesterol. The lateral diffusion coefficients (D) for supported lipid bilayer films of these layers without cholesterol were 7.9 ± 0.2, 7.9 ± 0.4 and 5.5 ± 0.1 μm2 s−1 respectively. The high fluidity reflected the super-hydrophilicity (contact angle of 0) of the ozone treated plasma glass substrate. Using DOPE conjugated Atto 655 as a probe, exposure of the lipid bilayer to a 30% wt/wt aqueous solution of PEG, followed by washing, dramatically increased the diffusion coefficients of the probe within the film. For example, the diffusion coefficient for the DOPC bilayer increases by nearly an order of magnitude to 51.4 ± 2.6 μm2 s−1. The autocorrelation curves for DOPC/DOPS (80/20 mol/mol) and DMPC bilayers required a two-component model for adequate fit of their behaviour yielding both fast and slow components of the diffusion. In all cases, when hydrophilic DOPE–Atto 655 was used as the probe, treatment of the lipid bilayer with PEG resulted in non-Brownian diffusion. Importantly, the observed diffusion behavior observed depends on the identity of probe. In contrast, when a hydrophobic probe (DOPE–NapthBodipy) was employed PEG showed relatively little impact on the observed diffusion rates. This was attributed to orientation of the reporter probe in the lipid bilayer and its aqueous interface. Specifically, DOPE–Atto 655 is believed to associate strongly with PEG mesh at the aqueous interface of the lipid bilayer, its diffusion strongly influenced by the structure in this region, whereas DOPE–NapthBodipy remains in the interior of the bilayer where it is relatively uninfluenced by PEG.

The interaction of two luminescent metallopolymers; [Ru(bpy)2(PVP)10]2+ and [Ru(bpy)2(CAIP)co-poly7]+, where bpy is 2,2′-bipyridyl, PVP is polyvinylpyridine, and (CAIP)co-poly7 is poly(styrene6-co-p-(aminomethyl)styrene) amide linked to 2-(4-carboxyphenyl)imidazo[4,5-f] [1,10]phenanthroline, with the Dawson polyoxomolybdate α-[Mo18O54(SO4)2]4− is described. Both metallopolymers undergo electrostatic association with the polyoxometalate. From both electronic and luminescence spectroscopy the thermodynamic products were determined to be {[Ru(bpy)2(PVP)10]4.5[Mo18O54(SO4)2]}5+ and {[Ru(bpy)2(CAIP)co-poly7]5[Mo18O54(SO4)2]}+, i.e. in both instances, the number of ruthenium centres in the cluster exceeds the number required for charge neutralization of the molybdate centre. Association quenches the luminescence of the metallopolymer although, consistent with the excess of Ru(II) present in the associated composites, emission is not completely extinguished even when a large excess of [Mo18O54(SO4)2]4− is present. The observed emission lifetime was not affected by [Mo18O54(SO4)2]4− therefore quenching was deemed static. The luminescent intensity data was found to fit best to a (sphere of action) Perrin model from which the radii of the quenching were calculated as 4.6 Å and 5.8 Å for [Ru(bpy)2(PVP)10]2+ and [Ru(bpy)2(CAIP co-poly)7]+ respectively. Both UV/Vis and resonance Raman data indicate the presence of a new optical transition centered around 490 nm for the composite, {[Ru(bpy)2(PVP)10]4.5[Mo18O54(SO4)2]}5+ but not for {[Ru(bpy)2(CAIP)co-poly7]5[Mo18O54(SO4)2]}+. This indicates strong electronic interaction between the metal centres in the former composite, which despite good thermodynamic analogy, is not observed for {[Ru(bpy)2(CAIP)co-poly7]5[Mo18O54(SO4)2]}+. These results are consistent with photoelectrochemical studies of layer by layer assemblies of these films which indicate that the ruthenium centre sensitizes polyoxometalate photo-oxidation of benzyl alcohol in {[Ru(bpy)2(PVP)10]4.5[Mo18O54(SO4)2]}5+ but not in {[Ru(bpy)2(CAIP)co-poly7]5[Mo18O54(SO4)2]}+.

Highly luminescent metallopolymers have been prepared from condensation reactions between the primary alkyl amines contained within a poly(styrene-p-(aminomethyl)styrene) polymer backbone and carboxylic acid groups on [Ru(bpy)2(CAIP)](ClO4)2, where CAIP is 2-(4-carboxyphenyl)imidazo[4,5-f][1,10]phenanthroline. The photophysical properties of these metallopolymers including emission and absorption spectroscopy, luminescent lifetimes and time-resolved emission anisotropy show strong solvent and pH dependence. These properties are rather insensitive to the loading of the metal centres on the polymer backbone suggesting weak electronic interactions between adjacent centres and the polymer backbone. The polymers form continuous films when drop-cast at an indium tin oxide interface and retain their strong luminescence. This processability and intense luminescence make them potentially useful for sensing and display applications.

Thin films of the adduct formed from the electrostatic association of the metallopolymer, [Ru(bpy)2(PVP)10]2+, and the Dawson polyoxomolybdate α-[Mo18O54(SO4)2]4–, POMo, have been formed on ITO electrodes using an alternate immersion approach. The Ru/POMo ratio is 4.5:1, which exceeds the 2:1 ratio expected on the basis of the charges of the Ru2+ and POMo4– building blocks. This behavior arises because of the polymeric character of the cation. In the presence of a substrate that has an abstractable proton such as benzyl alcohol, these ruthenium-sensitized polyoxomolybdate films generate significant photocurrents under visible irradiation. Significantly, increasing the surface coverage of the adduct from 1.4 × 10–10 to 8.1 × 10–10 mol cm–2 does not measurably increase the photocurrent observed. Scan-rate-dependent cyclic voltammetry reveals that the rate of homogeneous charge transport through the film is slow, which most likely results in only a fraction of the film thickness being active for photoelectrocatalysis. The photocurrent increases markedly when the driving force for the oxidation of POMo5–, created by the photoelectrocatalytic oxidation of benzyl alcohol, is increased. This result is consistent with the dynamics of heterogeneous electron transfer being centrally important to the regeneration of the photoelectrocatalyst. A system in which the surface coverage and applied overpotential are optimized produces a photocurrent density of 190 ± 18 nA cm–2 under 480 ± 5 nm irradiation.

The assembly of lipid bilayer membranes, using ultrasonic disruption of liposomes of L-α-Dimyristoyl phosphatidylcholine, across 820 nm diameter spherical cap gold cavity arrays is demonstrated.

A novel polymer based templated approach to creating highly ordered gold arrays with either positive or negative nano-scale features of controlled shape from truncated spheres to cuboids is presented.

Thin layers of an electrostatically associated adduct formed between the polyoxomolybdate, [S2Mo18O62]4−, and the metallopolymers [Ru(bpy)2(PVP)10]2+ or [Ru(bpy)2(CO-P PIC)7]2+ have been deposited onto electrodes using alternate immersion layer-by-layer assembly; PVP is poly(4-vinylpyridine), bpy is 2,2ʹ′-bipyridyl, CO-P is poly[(4-(aminomethyl)styrene)1 (4-vinylpyridine)6] and PIC is 2-(4-carboxyphenyl) imidazo [4,5-f][1,10] phenanthroline. Raman spectroscopy reveals that the Ru-PVP:POM films exhibit an additional vibrational mode at 900 cm− 1 that is not present in either of the components suggesting significant electronic communication between the ruthenium centres and the polyoxomolybdate. Despite the similarity of their redox and photonic properties, this optical transition is absent in the Ru-Co-P:POM layers. Significantly, the Ru-PVP:POM films generate a higher photocurrent (38 ± 1 nA cm− 2) than the Ru-Co-P:POM films (8.9 ± 0.8 nA cm− 2) or [S2Mo18O62]4− films (9.7 ± 1.1 nA cm− 2) under visible irradiation.► Metallopolymer-polyoxometallate films photocatalytically oxidise benzyl alcohol. ► Photocurrent intensity depends on new optical transition observed in Raman. ► Subtle changes in metallopolymer structure significantly affects photocurrent.

The spectroscopic and photophysical properties of [Ru(bpy)3]2[[Mo18O54(SO3)2], where bpy is 2,2′-bipyridyl and [Mo18O54(SO3)2]4− is either the α or β-sulfite containing polyoxomolybdate isomer, have been measured and compared with those for the well known but structurally distinct sulfate analogue, α-[Mo18O54(SO4)2]4−. Electronic difference spectroscopy revealed the presence of new spectral features around 480 nm, although they are weak in comparison with the [Ru(bpy)3]2[Mo18O54(SO4)2] analogue. Surprisingly, Stern–Volmer plots of [Ru(bpy)3]2+ luminescence quenching by the polyoxometallate revealed the presence of both static and dynamic quenching for both α and β-[Mo18O54(SO3)2]4−. The association constant inferred for the ion cluster [Ru(bpy)3]2α-[Mo18O54(SO4)2] is K = 5.9 ± 0.56 × 106 and that for [Ru(bpy)3]2β-[Mo18O54(SO4)2] is K = 1.0 ± 0.09 × 107. Unlike the sulfate polyoxometalates, both sulfite polyoxometalate–ruthenium adducts are non-luminescent. Despite the strong electrostatic association in the adducts resonance Raman and photoelectrochemical studies suggests that unlike the sulfato polyoxometalate analogue there is no sensitization of the polyoxometalate photochemistry by the ruthenium centre for the sulfite anions. In addition, the adducts exhibit photochemical lability in acetonitrile, attributable to decomposition of the ruthenium complex, which has not been observed for other [Ru(bpy)3]2+ -polyoxometalate adducts. These observations suggest that less electronic communication exists between the [Ru(bpy)3]2+ and the sulfite polyoxoanions relative to their sulfate polyoxoanion counterparts, despite their structural and electronic analogy. The main distinction between sulfate and sulfite polyoxometalates lies in their reversible reduction potentials, which are more positive by approximately 100 mV for the sulfite anions. This suggests that the capacity for [Ru(bpy)3]2+ or analogues to sensitize photoreduction in the adducts of polyoxometalates requires very sensitive redox tuning.

Nanosphere lithography has been used to create spherical recessed electrodes of nanometre dimensions capable of enhancing fluorescent signals.

Selective chemical modification of a gold nano-cavity array is achieved via nanoscale templating to create fibrinogen patterned cavities with a polyethylene glycol modified top surface. Application of a reducing potential to the array readily releases the protein from the cavities.

The capacity of ruthenium polypyridyl complexes as probes for combined confocal luminescence and resonance Raman imaging, enabled by their large Stokes shift, is demonstrated for a novel membrane sensitive Ru(II) polypyridyl peptide. Confocal luminescence and resonance Raman imaging provides complementary information about the membrane phospholipid regions of the cell and the location of the dye within the cell.

A mononuclear ruthenium(II) polypyridyl complex with an enlarged terpyridyl coordination cage was synthesized by the formal introduction of a carbon bridge between the coordinating pyridine rings. Structurally, the ruthenium(II) complex shows an almost perfect octahedral N6 coordination around the central RuII metal ion. The investigation of the photophysical properties reveals a triplet metal-to-ligand charge transfer emission with an unprecedented quantum yield of 13% and a lifetime of 1.36 μs at room temperature and in the presence of air oxygen. An exceptional small energy gap between light absorption and light emission, or Stokes shift, was detected. Additionally, time-dependent density functional theory calculations were carried out in order to characterize the ground state and both the singlet and triplet excited states. The exceptional properties of the new compound open the perspective of exploiting terpyridyl-like ruthenium complexes in photochemical devices under ambient conditions.

Monolayers of mono and bis-pyridine-substituted β- and γ-cyclodextrins have been self-assembled onto polycrystalline gold electrodes and electrochemically characterised. Surface enhanced Raman spectroscopy (SERS) has been used to probe the time dependent reorientation of the CDs and to probe the effect of co-adsorbing alkane thiols. Significantly, we observe novel SERS signatures for carbohydrate centred between 2100 and 2300 cm−1 which is sensitive to the orientation of the CD moiety on the gold surface. These modes are thought to arise from direct interactions between the CD pyranose ring and the gold substrate and are useful markers for assigning orientation of CD within a film. SERS confirmed, as was intimated from a previous electrochemical study, that significant reorientation of the CD groups within the film occurs on annealing and this orientation can be reversed by backfilling with alkane thiol. C60, in its unmodified state, was then immobilised through host–guest assembly into the backfilled CD monolayers to produce a 2:1 CD:C60 trilayer. These CD capped fullerenes are protected from photopolymerisation and exhibit well-defined redox processes.

The spectroscopic and photophysical properties of a synthetically versatile ruthenium complex [Ru(bpy)2(LH2)]2+ where LH2 is 2-(4-carboxyphenyl)imidazo[4,5-f][1,10]phenanthroline and bpy is 2,2-bipyridyl and its analogue, [Ru(bpy)2(LOMe)]2+ where the carboxyphenyl functionality is methylated are reported. Both complexes exhibit long-lived luminescence which for [Ru(bpy)2(LH2)]2+ is remarkably enhanced in aqueous compared to organic media. The pH dependence of the electronic absorption and emission spectra in water and acetonitrile are described and the influence of the protonation state of the 2-(4-carboxyphenyl)imidazo[4,5-f][1,10]phenanthroline ligand on the electronic structure of [Ru(bpy)2(LH2)]2+ is discussed. Oxidative quenching of the excited state of the complex by anthraquinone-2-carboxylic acid is investigated for both complexes. In polar media, this is a dynamic process suggesting that the quenching rate is controlled by bimolecular collision with a quenching rate constant, kq, of approximately 6.7 × 109 M−1 s−1 for [Ru(bpy)2(LH2)]2+. In contrast in aprotic solvent, dichloromethane, quenching occurs through a purely static mechanism indicating association between the luminophore and quencher, most likely through hydrogen bonding, between the carboxylic acid moieties of the ruthenium complex and the anthraquinone carboxylic derivative. The association constant for formation of the dyad was determined to be 565 L mol−1 in dichloromethane and the rate of electron transfer was estimated to be 4.7 × 107 s−1. By contrast, for the analogous complex in which the carboxylate is methyl protected mixed static and dynamic quenching behaviour in aprotic solvent.[Ru(bpy)2(LH2)]2+ where is LH2 is 2-(4-carboxyphenyl)imidazo[4,5-f][1,10]phenanthroline exhibits four protonation states whose photophysical properties are solvent dependent. Quenching of [Ru(bpy)2(LH2)]2+ by anthraquinone-2-carboxylic acid switches from a static unimolecular process in aprotic solvent to dynamic bimolecular quenching in aqueous medium. The static process is attributed to formation of a H-bonded dyad between donor and acceptor.

Two novel polyarginine labelled ruthenium polypyridyl dyes are reported, one conjugated to five, (Ru–Ahx–R5), and one to eight arginine residues, (Ru–Ahx–R8); both complexes exhibit long-lived, intense, and oxygen-sensitive luminescence; (Ru–R8) is passively, efficiently and very rapidly transported across the cell membrane into the cytoplasm without requirement for its permeablisation.

A highly reproducible and facile method for formation of ordered 2 dimensional arrays of CTAB protected 50 nm gold nanoparticles bonded to silicon wafers is described. The silicon wafers have been chemically modified with long-chain silanes terminated with thiol that penetrate the CTAB bilayer and chemically bind to the underlying gold nanoparticle. The silicon wafer provides a reproducibly smooth, chemically functionalizable and non-fluorescent substrate with a silicon phonon mode which may provide a convenient internal frequency and intensity calibration for vibrational spectroscopy. The CTAB bilayer provides a potentially biomimetic environment for analyte, yet allows a sufficiently small nanoparticle separation to achieve a significant electric field enhancement. The arrays have been characterized using SEM and Raman spectroscopy. These studies reveal that the reproducibility of the arrays is excellent both between batches (<10% RSD) and across a single batch (<5% RSD). The arrays also exhibit good stability, and the effect of temperature on the arrays was also investigated. The interaction of protein and amino acid with the nanoparticle arrays was investigated using Raman microscopy to investigate their potential in bio-SERS spectroscopy. Raman of phenylalanine and the protein bovine pancreatic trypsin inhibitor, BPTI were studied using 785 nm excitation, coincident with the surface plasmon absorbance of the array. The arrays exhibit SERS enhancements of the order of 2.6 × 104 for phenylalanine, the standard deviation on the relative intensity of the 1555 cm−1 mode of phenylalanine is less than 10% for 100 randomly distributed locations across a single substrate and less than 20% between different substrates. Significantly, comparisons of the Raman spectra of the protein and phenylalanine in solution and immobilized on the nanoparticle arrays indicates that the protein is non-randomly orientated on the arrays. Selective SERS enhancements suggest that aromatic residues penetrate through the bilayer inducing conformational changes in the protein.

A novel photoactive complex, [Ru-LH]2+, comprising a ruthenium bisbipyridyl centre coordinated to 2-(4-adamantylphenyl)imidazo[4,5-f] [1,10]phenanthroline (LH) was prepared from the parent complex, [Ru(bpy)2(CAIP)]2+, where CAIP is 2-(4-carboxyphenyl)imidazo[4,5-f] [1,10]phenanthroline. The complex exhibits an intense luminescence and both its absorbance and emission properties are pH dependent. This pH dependence is associated with two ionisable sites at the imidazole on the heteroligand, for which pKas of 1.66 and 8.5 were determined from UV–Vis spectroscopy. The photophysics and resonance Raman spectroscopy of the complex as a function of pH are presented. Whereas the energy of the metal to ligand charge transfer transition is strongly influenced by pH, the pKa∗ suggests the lowest energy emitting state remains on the bipyridyl moiety regardless of pH. The self-assembly of the ruthenium complex with both native β-cyclodextrin and β-cyclodextrin covalently linked to a viologen acceptor, mono-6-(1-alkyl-4,4′-bipyridino)-β-cyclodextrin, CD-MV2+, was studied using 1H NMR spectroscopy. Assembly is driven by a hydrophobic host–guest interaction between the adamantyl pendant and the cyclodextrin. The association constant for self-assembly with β-cyclodextrin was determined to be 2.9 (±0.5) × 104 M−1, independent of the pH of the media. However, association with CD-MV2+ was found to be pH dependent, with Kassoc in neutral media determined to be 8.8 (±0.2) × 103 M−1 compared with 2.24 (±0.2) × 104 M−1 at pH 10. The photophysics of the assembly indicated that photoinduced electron transfer (PET) between the ruthenium centre and the pendant methyl viologen terminus is strongly and reversibly modulated by pH. Between pH 4 and 7, photoinduced electron transfer is, at best, very inefficient. On the other hand, at pHs exceeding 8.5, corresponding to complete deprotonation of the benzimidazole, an efficient electron transfer occurs and the resulting MV+ radical was identified in both transient absorbance studies and steady state photolysis. The origins of this pH-modulated electron transfer process are discussed.A novel photoactive supramolecular assembly is described wherein a ruthenium bisbipyridyl centre with an adamantyl pendant is included in the β-cyclodextrin functionalised with a viologen acceptor. Photo induced electron transfer from the ruthenium to viologen is demonstrated to occur. The rate of this process is by modulated by protonation/deprotonation of the benzimidazole bridge.

Voltammetric, photo-physical and photo-electrochemical properties of the Dawson polyoxometalate anions α-[S2M18O62]4− (M = Mo, W) are presented, both in the presence and absence of a series of [RuIILn]+/2+ cations [Ln = (bpy)3, (bpy)2(Im)2, (bpy)2(dpq), (bpy)2(box) and (biq)2(box)]. Electrochemical processes for both the anion and RuII/III couples were detected in solutions of the salts [RuIILn]2[S2M18O62] in dimethylformamide (0.1 M Bu4NPF6) by both cyclic and hydrodynamic voltammetries. Responses were also detected when the solid salts were adhered to the surface of a glassy carbon electrode in contact with an electrolyte in which they are insoluble (CH3CN; 0.1M Bu4NPF6). Photolysis experiments were performed on solutions of the salts [R4N]4[S2M18O62] (R = n-butyl or n-hexyl) and [RuIILn]2[S2M18O62] at 355 and 420 nm in dimethylformamide and acetonitrile in the presence and absence of benzyl alcohol (10% v/v). When associated with [Ru(bpy)3]2+, the molybdate anion exhibited a large increase in the quantum yield for photo-reduction at 420 nm. The quantum yield for the tungstate analogue was lower but the experiments again provided clear evidence for sensitization of the photo-reduction reaction in the visible spectral region. The origin of this sensitization is ascribed to the new optical transition observed around 480 nm in static ion clusters {[Ru(bpy)3][S2M18O62]}2− and {[Ru(bpy)3]2[S2M18O62]} present in solution. Measurable photocurrents resulted from irradiation of solutions of the anions with white light in the presence of the electron donor dimethylformamide. Evidence is also presented for possible quencher–fluorophore interactions in the presence of certain [RuIILn]+ cations.

The synthesis, spectroscopic and electrochemical characterisation of a series of optically tuneable, ruthenium (II) and osmium (II) polypyridyl complexes, O,N coordinated to electroactive donor ligand, bis-2,5-(2-benzoxazolyl)-hydroquinone (bbhq) is described. The complexes exhibit a rich optical spectroscopy which can be controlled through the redox state of the metal and bbhq ligand. The influence of both the metal and counter-ligand identity on the optical properties of these hydroquinone-based complexes is addressed.Regardless of the identity of metal or counter-ligand, it is the bbhq which is the site of the most facile oxidation and hydroquinone, semiquinone (bbsq) and quinone (bbq) can be generated electrochemically. In each instance, the semiquinone is strongly stabilised with respect to disproportionation, reflected in large stability constants for this moiety. The levels of orbital mixing between metal and ligand are discussed on the basis of the optical properties of the complex and the nature of the metal and counter-ligand. In addition, we address, for the first time, the effect of metal and counter-ligand on the photostability, of Ru(II) and Os(II) hydroquinone bound complexes. We find that like other ruthenium (II) complexes containing strong σ-bonding ligands, the M(bpy)2 containing complexes are photostable, but the [Ru(biq)2(bbhq)]+ complex is relatively photolabile.Ruthenium and osmium polypyridyl complexes coordinated to bis-2,5-(2-benzoxazolyl)-hydroquinone exhibit a rich potential dependent optical spectroscopy. The semiquinone state is strongly stabilised towards disproportionation in all complexes and this stability and photochemical stability is strongly dependent on the identity of the metal and ancillary ligand.

The synthesis and a detailed investigation into the electronic properties of mononuclear and dinuclear Ru(II) complexes of the ligand bis(2-hydroxyphenyl)-2,5-dihydropyrazine (H2BHD) is described. In these complexes the Ru(II) moieties are bound through O,N coordination to an anionic phenolate and the pyrazine bridge. Relatively few reports are available on the dinuclear complexes bridged across a phenolate and this study provides an opportunity to examine the impact of reduced oxygen donor ligands on metal–metal communication. The results presented here reveal some very unusual behavior whereby the apparent location of the LUMO changes between the mononuclear and dinuclear complexes. The lowest energy optical transition appears to involve the peripheral bipyridine ligand as acceptor in the mononuclear complex, whereas this ligand is not involved in the lowest energy optical transition in the dinuclear complex. The origin of this difference is not clear, however, significant changes in the electronic properties of the mononuclear complex are observed on coordination of the second metal, reflected in significant alterations in the electrochemistry of the bridge and metals as well as changes in the optical spectroscopy. The BHD2− bridge is shown to support weakly coupled class II behavior according to the Robin and Day classification, reflected in a Kc of 335.

Supported Lipid Bilayers (SLBs) are versatile models capable of mimicking some of the key properties of the cell membrane, including for example lipid fluidity, domain formation and protein support, without the challenging complexity of the real biological system. This is important both from the perspective of understanding the behaviour and role of the lipid membrane in cell structure and signalling, as well as in development of applications of lipid membranes across domains as diverse as sensing and drug delivery. Lipid and protein diffusion within the membrane is vital to its function and there are several key experimental methods used to study membrane dynamics. Amongst the optical methods are Fluorescence Recovery After Photobleaching (FRAP), single particle tracking and Fluorescence Correlation (and Fluorescence Lifetime Correlation) Spectroscopy (FCS/FLCS). Each of these methods can provide different and often complementary perspectives on the dynamics of the fluid membrane. Although FCS is well established, FLCS is a relatively new technique and both methods have undergone a number of extensions in recent years which improve their precision and accuracy in studying supported lipid bilayers, most notably z-scan methods. This short review focusses on FCS and FLCS and their recent applications, specifically to artificial lipid bilayer studies addressing key issues of cell membrane behaviour.Download full-size image

Fluorescence microscopy has undergone a dramatic evolution over the past two decades with development of super-resolution far-field microscopy methods that break the light diffraction limited resolution of conventional microscopy, offering unprecedented opportunity to interrogate cellular processes at the nanoscale. However, these methods make special demands of the luminescent agents used for contrast and development of probes suited to super-resolution fluorescent methods is still relatively in its infancy. In spite of their many photophysical advantages, metal complex luminophores have not yet been considered as probes in this regard, where to date, only organic fluorophores have been applied. Here, we report the first examples of metal complex luminophores applied as probes for use in stimulated emission depletion (STED) microscopy. Exemplified with endoplasmic reticulum and nuclear targeting complexes we demonstrate that luminescent Ru(II) polypyridyl complexes can, through signal peptide targeting, be precisely and selectively delivered to key cell organelles without the need for membrane permeabilization, to give high quality STED images of these organelles. Detailed features of the tubular ER structure are revealed and in the case of the nuclear targeting probe we exploit the molecular light switch properties of a dipyrido[3,2-a:2′,3′-c]phenazine containing complex which emits only on DNA/RNA binding to give outstanding STED contrast and resolution of the chromosomes within the nucleus. Comparing performance with a member of the AlexaFluor family commonly recommended for STED, we find that the performance of the ruthenium complexes is superior across both CW and gated STED microscopy methods in terms of image resolution and photostability. The large Stokes shifts of the Ru probes permit excellent matching of the stimulating depletion laser with their emission whilst avoiding anti-Stokes excitation. Their long lifetimes make them particularly amenable to gated STED, giving a much wider window for gating than traditional probes. Our findings indicate that ruthenium polypyridyl peptide targeted probes are a powerful new partner to STED microscopy, opening up new approaches to probe design for STED microscopy.

A highly reproducible and facile method for formation of ordered 2 dimensional arrays of CTAB protected 50 nm gold nanoparticles bonded to silicon wafers is described. The silicon wafers have been chemically modified with long-chain silanes terminated with thiol that penetrate the CTAB bilayer and chemically bind to the underlying gold nanoparticle. The silicon wafer provides a reproducibly smooth, chemically functionalizable and non-fluorescent substrate with a silicon phonon mode which may provide a convenient internal frequency and intensity calibration for vibrational spectroscopy. The CTAB bilayer provides a potentially biomimetic environment for analyte, yet allows a sufficiently small nanoparticle separation to achieve a significant electric field enhancement. The arrays have been characterized using SEM and Raman spectroscopy. These studies reveal that the reproducibility of the arrays is excellent both between batches (<10% RSD) and across a single batch (<5% RSD). The arrays also exhibit good stability, and the effect of temperature on the arrays was also investigated. The interaction of protein and amino acid with the nanoparticle arrays was investigated using Raman microscopy to investigate their potential in bio-SERS spectroscopy. Raman of phenylalanine and the protein bovine pancreatic trypsin inhibitor, BPTI were studied using 785 nm excitation, coincident with the surface plasmon absorbance of the array. The arrays exhibit SERS enhancements of the order of 2.6 × 104 for phenylalanine, the standard deviation on the relative intensity of the 1555 cm−1 mode of phenylalanine is less than 10% for 100 randomly distributed locations across a single substrate and less than 20% between different substrates. Significantly, comparisons of the Raman spectra of the protein and phenylalanine in solution and immobilized on the nanoparticle arrays indicates that the protein is non-randomly orientated on the arrays. Selective SERS enhancements suggest that aromatic residues penetrate through the bilayer inducing conformational changes in the protein.

Two novel polyarginine labelled ruthenium polypyridyl dyes are reported, one conjugated to five, (Ru–Ahx–R5), and one to eight arginine residues, (Ru–Ahx–R8); both complexes exhibit long-lived, intense, and oxygen-sensitive luminescence; (Ru–R8) is passively, efficiently and very rapidly transported across the cell membrane into the cytoplasm without requirement for its permeablisation.

The targeted delivery of luminescent Ru(II) polypyridyl complexes to the nucleus of live mammalian cells by a Nuclear Localisation Signal (NLS) peptide; NF-κB is demonstrated.

A ruthenium(II) polypyridyl–BODIPY dyad is presented which exhibits a solvent switchable dual emission. Intense oxygen sensitive emission from the ruthenium centre and O2 independent emission from the BODIPY centre, are both observed in organic media. In aqueous media, the BODIPY emission is reversibly switched off leaving only a ruthenium centred emission. The materials are interesting both as self-referenced O2 probes and for cell/tissue imaging.

Blood platelet adhesion is crucial in dictating haemocompatibility of medical implants and in platelet capture in diagnostics. Understanding the role of platelet activation in dictating platelet adhesion at chemically modified interfaces is important but relatively unexplored. Using scanning electron microscopy and confocal fluorescence microscopy a quantitative assessment of capture of blood platelets at self-assembled monolayers and mixed monolayers (SAMs) on gold as a function of the activation status of the platelets was conducted. Single and mixed monolayers were prepared using thiol-functionalized arginine-glycine-aspartic acid (RGD), C-Ahx-GRGDS (Ahx = aminohexanoic acid linker), thiolated poly(ethylene)glycol (PEG-COOH) and 1-octanethiol. When incubated with suspensions of resting platelets, RGD promoted platelet adhesion compared to bare or alkanethiol modified gold. Increasing the alkanethiol ratio in the deposition solution decreased the extent of platelet adhesion. Platelet adhesion increased approximately 3 fold at PEG-COO- modified surfaces compared to RGD-alone. Platelets adhered to RGD or mixed RGD:alkane SAM surfaces were found to be captured in their resting state. In contrast, platelets captured at PEG-COO- SAM surfaces were activated by these substrates. The effect of treating platelets with the chemical activators, Mn2+ or DTT or the physiological activator, thrombin, on the capture efficiency and activation at RGD modified surfaces was also investigated. Mn2+ treated platelets presented similar adhesion to untreated platelets, while surprisingly DTT yielded a very significant decrease in platelet adhesion. And, any platelets that were captured, were in a resting state. Thrombin activated platelets were captured with similar efficiencies as untreated platelets. However, the platelets captured were fully activated. The distinction between capture of chemically and physiologically activated platelet is interesting and likely to originate from differences in the conformation of the integrin induced by each process. Finally, platelet adhesion to each surface could be reversed by incubation with a solution of linear or cyclical RGD or PEG-COO- for the RGD and PEGCOO- surfaces respectively. The specificity of platelet removal confirmed that platelet adhesion at RGD surfaces is occurring through integrin–RGD interactions.

A novel Near Infra-Red emitting BODIPY derivative is presented which exhibits the largest Stokes shift thus far reported for a BODIPY compound.

A novel polymer based templated approach to creating highly ordered gold arrays with either positive or negative nano-scale features of controlled shape from truncated spheres to cuboids is presented.

The assembly of lipid bilayer membranes, using ultrasonic disruption of liposomes of L-α-Dimyristoyl phosphatidylcholine, across 820 nm diameter spherical cap gold cavity arrays is demonstrated.

Monolayers of mono and bis-pyridine-substituted β- and γ-cyclodextrins have been self-assembled onto polycrystalline gold electrodes and electrochemically characterised. Surface enhanced Raman spectroscopy (SERS) has been used to probe the time dependent reorientation of the CDs and to probe the effect of co-adsorbing alkane thiols. Significantly, we observe novel SERS signatures for carbohydrate centred between 2100 and 2300 cm−1 which is sensitive to the orientation of the CD moiety on the gold surface. These modes are thought to arise from direct interactions between the CD pyranose ring and the gold substrate and are useful markers for assigning orientation of CD within a film. SERS confirmed, as was intimated from a previous electrochemical study, that significant reorientation of the CD groups within the film occurs on annealing and this orientation can be reversed by backfilling with alkane thiol. C60, in its unmodified state, was then immobilised through host–guest assembly into the backfilled CD monolayers to produce a 2:1 CD:C60 trilayer. These CD capped fullerenes are protected from photopolymerisation and exhibit well-defined redox processes.

The interaction of two luminescent metallopolymers; [Ru(bpy)2(PVP)10]2+ and [Ru(bpy)2(CAIP)co-poly7]+, where bpy is 2,2′-bipyridyl, PVP is polyvinylpyridine, and (CAIP)co-poly7 is poly(styrene6-co-p-(aminomethyl)styrene) amide linked to 2-(4-carboxyphenyl)imidazo[4,5-f] [1,10]phenanthroline, with the Dawson polyoxomolybdate α-[Mo18O54(SO4)2]4− is described. Both metallopolymers undergo electrostatic association with the polyoxometalate. From both electronic and luminescence spectroscopy the thermodynamic products were determined to be {[Ru(bpy)2(PVP)10]4.5[Mo18O54(SO4)2]}5+ and {[Ru(bpy)2(CAIP)co-poly7]5[Mo18O54(SO4)2]}+, i.e. in both instances, the number of ruthenium centres in the cluster exceeds the number required for charge neutralization of the molybdate centre. Association quenches the luminescence of the metallopolymer although, consistent with the excess of Ru(II) present in the associated composites, emission is not completely extinguished even when a large excess of [Mo18O54(SO4)2]4− is present. The observed emission lifetime was not affected by [Mo18O54(SO4)2]4− therefore quenching was deemed static. The luminescent intensity data was found to fit best to a (sphere of action) Perrin model from which the radii of the quenching were calculated as 4.6 Å and 5.8 Å for [Ru(bpy)2(PVP)10]2+ and [Ru(bpy)2(CAIP co-poly)7]+ respectively. Both UV/Vis and resonance Raman data indicate the presence of a new optical transition centered around 490 nm for the composite, {[Ru(bpy)2(PVP)10]4.5[Mo18O54(SO4)2]}5+ but not for {[Ru(bpy)2(CAIP)co-poly7]5[Mo18O54(SO4)2]}+. This indicates strong electronic interaction between the metal centres in the former composite, which despite good thermodynamic analogy, is not observed for {[Ru(bpy)2(CAIP)co-poly7]5[Mo18O54(SO4)2]}+. These results are consistent with photoelectrochemical studies of layer by layer assemblies of these films which indicate that the ruthenium centre sensitizes polyoxometalate photo-oxidation of benzyl alcohol in {[Ru(bpy)2(PVP)10]4.5[Mo18O54(SO4)2]}5+ but not in {[Ru(bpy)2(CAIP)co-poly7]5[Mo18O54(SO4)2]}+.

A first investigation into the application of a luminescent osmium(II) bipyridine complex to live cell imaging is presented. Osmium(II) (bis-2,2-bipyridyl)-2(4-carboxylphenyl) imidazo[4,5f][1,10]phenanthroline was prepared and conjugated to octaarginine, a cell penetrating peptide. The photophysics, cell uptake and cytotoxicity of this osmium complex conjugate were performed and compared with its ruthenium analogue. Cell uptake and distribution of both ruthenium and osmium conjugates were very similar with rapid transmembrane transport of the osmium probe (complete within approx. 20 min) and dispersion throughout the cytoplasm and organelles. The near-infrared (NIR) emission of the osmium complex (λmax 726 nm) coincides well with the biological optical window and this facilitated luminescent and luminescence lifetime imaging of the cell which was well resolved from cell autofluorescence. The large Stokes shift of the emission also permitted resonance Raman mapping of the dye within CHO cells. Rather surprisingly, the osmium conjugate exhibited very low cytotoxicity when incubated both in the dark and under visible irradiation. This was attributed to the remarkable stability of this complex which was reflected by the complete absence of photo-bleaching of the complex even under extended continuous irradiation. In addition, when compared to its ruthenium analogue its luminescence was short-lived in water therefore rendering it insensitive to O2.

Selective chemical modification of a gold nano-cavity array is achieved via nanoscale templating to create fibrinogen patterned cavities with a polyethylene glycol modified top surface. Application of a reducing potential to the array readily releases the protein from the cavities.

The capacity of ruthenium polypyridyl complexes as probes for combined confocal luminescence and resonance Raman imaging, enabled by their large Stokes shift, is demonstrated for a novel membrane sensitive Ru(II) polypyridyl peptide. Confocal luminescence and resonance Raman imaging provides complementary information about the membrane phospholipid regions of the cell and the location of the dye within the cell.

The spectroscopic and photophysical properties of [Ru(bpy)3]2[[Mo18O54(SO3)2], where bpy is 2,2′-bipyridyl and [Mo18O54(SO3)2]4− is either the α or β-sulfite containing polyoxomolybdate isomer, have been measured and compared with those for the well known but structurally distinct sulfate analogue, α-[Mo18O54(SO4)2]4−. Electronic difference spectroscopy revealed the presence of new spectral features around 480 nm, although they are weak in comparison with the [Ru(bpy)3]2[Mo18O54(SO4)2] analogue. Surprisingly, Stern–Volmer plots of [Ru(bpy)3]2+ luminescence quenching by the polyoxometallate revealed the presence of both static and dynamic quenching for both α and β-[Mo18O54(SO3)2]4−. The association constant inferred for the ion cluster [Ru(bpy)3]2α-[Mo18O54(SO4)2] is K = 5.9 ± 0.56 × 106 and that for [Ru(bpy)3]2β-[Mo18O54(SO4)2] is K = 1.0 ± 0.09 × 107. Unlike the sulfate polyoxometalates, both sulfite polyoxometalate–ruthenium adducts are non-luminescent. Despite the strong electrostatic association in the adducts resonance Raman and photoelectrochemical studies suggests that unlike the sulfato polyoxometalate analogue there is no sensitization of the polyoxometalate photochemistry by the ruthenium centre for the sulfite anions. In addition, the adducts exhibit photochemical lability in acetonitrile, attributable to decomposition of the ruthenium complex, which has not been observed for other [Ru(bpy)3]2+ -polyoxometalate adducts. These observations suggest that less electronic communication exists between the [Ru(bpy)3]2+ and the sulfite polyoxoanions relative to their sulfate polyoxoanion counterparts, despite their structural and electronic analogy. The main distinction between sulfate and sulfite polyoxometalates lies in their reversible reduction potentials, which are more positive by approximately 100 mV for the sulfite anions. This suggests that the capacity for [Ru(bpy)3]2+ or analogues to sensitize photoreduction in the adducts of polyoxometalates requires very sensitive redox tuning.

Nanosphere lithography has been used to create spherical recessed electrodes of nanometre dimensions capable of enhancing fluorescent signals.