Blue Electrogenerated Chemiluminescence from Water-Soluble Iridium Complexes Containing Sulfonated Phenylpyridine or Tetraethylene Glycol Derivatized Triazolylpyridine Ligands.

Incorporating phenylpyridine- and triazolylpyridine-based ligands decorated with methylsulfonate or tetraethylene glycol (TEG) groups, a series of iridium(III) complexes has been created for green and blue electrogenerated chemiluminescence under analytically useful aqueous conditions, with tri-n-propylamine as a coreactant. The relative electrochemiluminescence (ECL) intensities of the complexes were dependent on the sensitivity of the photodetector over the wavelength range and the pulse time of the applied electrochemical potential. In terms of the integrated area of corrected ECL spectra, with a pulse time of 0.5 s, the intensities of the Ir(III) complexes were between 18 and 102 % that of [Ru(bpy)3 ](2+) (bpy=2,2'-bipyridine). However, when the intensities were measured with a typical bialkali photomultiplier tube, the signal of the most effective blue emitter, [Ir(df-ppy)2 (pt-TEG)](+) (df-ppy=2-(2,4-difluorophenyl)pyridine anion, pt-TEG=1-(2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl)-4-(2-pyridyl)-1,2,3-triazole), was over 1200 % that of the orange-red emitter [Ru(bpy)3 ](2+) . A combined experimental and theoretical investigation of the electrochemical and spectroscopic properties of the Ir(III) complexes indicated that the greater intensity from [Ir(df-ppy)2 (pt-TEG)](+) relative to those of the other Ir(III) complexes resulted from a combination of many factors, rather than being significantly favored in one area.

Electrogenerated chemiluminescence of tris(2,2' bipyridine)ruthenium(II) using common biological buffers as co-reactant, pH buffer and supporting electrolyte.

A series of aliphatic tertiary amines (HEPES, POPSO, EPPS and BIS-TRIS) commonly used to buffer the pH in biological experiments, were examined as alternative, non-toxic co-reactants for the electrogenerated chemiluminescence (ECL) of tris(2,2'-bipyridine)ruthenium(ii) ([Ru(bpy)3](2+)). These were found to be very attractive as "multi-tasking" reagents, serving not only as co-reactants, but also fulfiling the roles of pH buffer and supporting electrolyte within an aqueous environment; thus significantly simplifying the overall ECL analysis. Sub-nanomolar detection limits were obtained for [Ru(bpy)3](2+) in the presence of BIS-TRIS, making this species an valuable option for co-reactant ECL-based bioanalytical applications.

Red-green-blue electrogenerated chemiluminescence utilizing a digital camera as detector.

Exploiting the distinct excitation and emission properties of concomitant electrochemiluminophores in conjunction with the inherent color selectivity of a conventional digital camera, we create a new strategy for multiplexed electrogenerated chemiluminescence detection, suitable for the development of low-cost, portable clinical diagnostic devices. Red, green and blue emitters can be efficiently resolved over the three-dimensional space of ECL intensity versus applied potential and emission wavelength. As the relative contribution ratio of each emitter to the photographic RGB channels is constant, the RGB ECL intensity versus applied-potential curves could be effectively isolated to a single emitter at each potential.

Control of excitation and quenching in multi-colour electrogenerated chemiluminescence systems through choice of co-reactant.

We demonstrate a new approach to manipulate the selective emission in mixed electrogenerated chemiluminescence (ECL) systems, where subtle changes in co-reactant properties are exploited to control the relative electron-transfer processes of excitation and quenching. Two closely related tertiary-amine co-reactants, tri-n-propylamine and N,N-diisopropylethylamine, generate remarkably different emission profiles: one provides distinct green and red ECL from [Ir(ppy)3] (ppy=2-phenylpyridinato-C2,N) and a [Ru(bpy)3](2+) (bpy=2,2'-bipyridine) derivative at different applied potentials, whereas the other generates both emissions simultaneously across a wide potential range. These phenomena can be rationalized through the relative exergonicities of electron-transfer quenching of the excited states, in conjunction with the change in concentration of the quenchers over the applied potential range.

Understanding electrogenerated chemiluminescence efficiency in blue-shifted iridium(III)-complexes: an experimental and theoretical study.

Compared to tris(2-phenylpyridine)iridium(III) ([Ir(ppy)3 ]), iridium(III) complexes containing difluorophenylpyridine (df-ppy) and/or an ancillary triazolylpyridine ligand [3-phenyl-1,2,4-triazol-5-ylpyridinato (ptp) or 1-benzyl-1,2,3-triazol-4-ylpyridine (ptb)] exhibit considerable hypsochromic shifts (ca. 25-60 nm), due to the significant stabilising effect of these ligands on the HOMO energy, whilst having relatively little effect on the LUMO. Despite their lower photoluminescence quantum yields compared with [Ir(ppy)3 ] and [Ir(df-ppy)3 ], the iridium(III) complexes containing triazolylpyridine ligands gave greater electrogenerated chemiluminescence (ECL) intensities (using tri-n-propylamine (TPA) as a co-reactant), which can in part be ascribed to the more energetically favourable reactions of the oxidised complex (M(+) ) with both TPA and its neutral radical oxidation product. The calculated iridium(III) complex LUMO energies were shown to be a good predictor of the corresponding M(+) LUMO energies, and both HOMO and LUMO levels are related to ECL efficiency. The theoretical and experimental data together show that the best strategy for the design of efficient new blue-shifted electrochemiluminophores is to aim to stabilise the HOMO, while only moderately stabilising the LUMO, thereby increasing the energy gap but ensuring favourable thermodynamics and kinetics for the ECL reaction. Of the iridium(III) complexes examined, [Ir(df-ppy)2 (ptb)](+) was most attractive as a blue-emitter for ECL detection, featuring a large hypsochromic shift (λmax =454 and 484 nm), superior co-reactant ECL intensity than the archetypal homoleptic green and blue emitters: [Ir(ppy)3 ] and [Ir(df-ppy)3 ] (by over 16-fold and threefold, respectively), and greater solubility in polar solvents.

Chemiluminescence detection with water-soluble iridium(III) complexes containing a sulfonate-functionalised ancillary ligand.

The chemiluminescence from four cyclometalated iridium(III) complexes containing an ancillary bathophenanthroline-disulfonate ligand exhibited a wide range of emission colours (green to red), and in some cases intensities that are far greater than the commonly employed benchmark reagent, [Ru(bpy)3](2+). A similar complex incorporating a sulfonated triazolylpyridine-based ligand enabled the emission to be shifted into the blue region of the spectrum, but the responses with this complex were relatively poor. DFT calculations of electronic structure and emission spectra support the experimental findings.

Electrochemiluminescent ruthenium(II) N-heterocyclic carbene complexes: a combined experimental and theoretical study.

A series of four Ru(II) complexes of the form [Ru(bpy)2(C(^)N)](2+) (where C(^)N is a bidentate pyridine-functionalized imidazolylidene- or benzimidazolylidene-based N-heterocyclic carbene (NHC) ligand and bpy is 2,2'-bipyridine) have been synthesized using a Ag(I) transmetalation protocol from the Ru(II) precursor compound, Ru(bpy)2Cl2. The synthesized azolium salts and Ru(II) complexes were characterized by elemental analysis, (1)H and (13)C NMR spectroscopy, cyclic voltammetry, and electronic absorption and emission spectroscopy. The molecular structures for two benzimidazolium salts and three Ru(II) complexes were determined by single crystal X-ray diffraction. The complexes display photoluminescence within the range 611-629 nm, with the emission wavelength of the benzimidazolylidene containing structures, slightly blue-shifted relative to the imidazolylidene containing complexes. All complexes exhibited a reversible, one-electron oxidation, which is assigned to the Ru(2+/3+) redox couple. When compared to [Ru(bpy)3](2+), complexes of imidazolylidene containing ligands were oxidized at more negative potentials, while those of the benzimidazolylidene containing ligands were oxidized at more positive potentials. All four complexes exhibited moderately intense electrochemiluminescence (ECL) with the obtained ECL spectra closely resembling the photoluminescence spectra. The ability to predictably fine-tune the highest occupied molecular orbital (HOMO) level of the Ru(II) complexes via the flexible synthetic strategy offered by NHCs is valuable for the design of ECL-based multiplexed detection strategies.

Chemiluminescence from osmium(II) complexes with phenanthroline, diphosphine and diarsine ligands.

The reaction of various [Os(L)(2)(L')](2+) complexes (where L and L' are phenanthroline, diphosphine or diarsine ligands) and organic reducing agents after chemical or electrochemical oxidation of the reactants produces an emission of light corresponding to MLCT transitions. In certain instances, the emission was greater than that of [Ru(bipy)(3)](2+), but the relative signals were dependent on many factors, including reagent concentration, mode of oxidation, reducing agent and the sensitivity of the photodetector over the wavelength range.

Electrochemiluminescent monomers for solid support syntheses of Ru(II)-PNA bioconjugates: multimodal biosensing tools with enhanced duplex stability.

The feasibility of devising a solid support mediated approach to multimodal Ru(II)-peptide nucleic acid (PNA) oligomers is explored. Three Ru(II)-PNA-like monomers, [Ru(bpy)(2)(Cpp-L-PNA-OH)](2+) (M1), [Ru(phen)(2)(Cpp-L-PNA-OH)](2+) (M2), and [Ru(dppz)(2)(Cpp-L-PNA-OH)](2+) (M3) (bpy = 2,2'-bipyridine, phen = 1,10-phenanthroline, dppz = dipyrido[3,2-a:2',3'-c]phenazine, Cpp-L-PNA-OH = [2-(N-9-fluorenylmethoxycarbonyl)aminoethyl]-N-[6-(2-(pyridin-2yl)pyrimidine-4-carboxamido)hexanoyl]-glycine), have been synthesized as building blocks for Ru(II)-PNA oligomers and characterized by IR and (1)H NMR spectroscopy, mass spectrometry, electrochemistry and elemental analysis. As a proof of principle, M1 was incorporated on the solid phase within the PNA sequences H-g-c-a-a-t-a-a-a-a-Lys-NH(2) (PNA1) and H-P-K-K-K-R-K-V-g-c-a-a-t-a-a-a-a-lys-NH(2) (PNA4) to give PNA2 (H-g-c-a-a-t-a-a-a-a-M1-lys-NH(2)) and PNA3 (H-P-K-K-K-R-K-V-g-c-a-a-t-a-a-a-a-M1-lys-NH(2)), respectively. The two Ru(II)-PNA oligomers, PNA2 and PNA3, displayed a metal to ligand charge transfer (MLCT) transition band centered around 445 nm and an emission maximum at about 680 nm following 450 nm excitation in aqueous solutions (10 mM PBS, pH 7.4). The absorption and emission response of the duplexes formed with the cDNA strand (DNA: 5'-T-T-T-T-T-T-T-A-T-T-G-C-T-T-T-3') showed no major variations, suggesting that the electronic properties of the Ru(II) complexes are largely unaffected by hybridization. The thermal stability of the PNA·DNA duplexes, as evaluated from UV melting experiments, is enhanced compared to the corresponding nonmetalated duplexes. The melting temperature (T(m)) was almost 8 °C higher for PNA2·DNA duplex, and 4 °C for PNA3·DNA duplex, with the stabilization attributed to the electrostatic interaction between the cationic residues (Ru(II) unit and positively charged lysine/arginine) and the polyanionic DNA backbone. In presence of tripropylamine (TPA) as co-reactant, PNA2, PNA3, PNA2·DNA and PNA3·DNA displayed strong electrochemiluminescence (ECL) signals even at submicromolar concentrations. Importantly, the combination of spectrochemical, thermal and ECL properties possessed by the Ru(II)-PNA sequences offer an elegant approach for the design of highly sensitive multimodal biosensing tools.

A simple, low-cost instrument for electrochemiluminescence immunoassays based on a Raspberry Pi and screen-printed electrodes.

A powerful, yet low-cost and semi-portable electrochemiluminescence (ECL) biosensing device is described. It is constructed around a Raspberry Pi single-board computer, which serves as the controller and user interface. The Pi is interfaced with an expansion board that controls the potential applied to a disposable screen-printed electrode and facilitates data acquisition from a photomultiplier tube (PMT), which detects the ECL emission from the sensor surface. As proof-of-concept, we demonstrate that this arrangement can quantitate tris(2,2'-bipyridine)ruthenium(II) ([Ru(bpy)3]2+]) with an estimated limit of detection (LOD) of 20 pM, and C-reactive protein with an LOD of 50 fg mL-1. The analytical performance of the Raspberry Pi-based setup is comparable to a conventional ECL configuration (computer, potentiostat and photodetector). The Raspberry Pi-based setup can replace a conventional ECL setup, at a fraction of the cost, without sacrificing sensitivity or versatility. The combination of a single-board computer and a sensitive light detector represents a significant step towards translating ECL instruments into mobile, point-of-care diagnostic platforms.

Highly stable ECL active films formed by the electrografting of a diazotized ruthenium complex generated in situ from the amine.

The electrodeposition of the electrochemiluminescent (ECL) ruthenium complex, bis(2,2'-bipyridyl)(4'-(4-aminophenyl)-2,2'-bipyridyl)ruthenium(II), [Ru(bpy)(2)(apb)](2+), via the in situ formation of a diazonium species from aqueous media is reported. Surface characterization undertaken using X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) determined that the layer is bound to the substrate via azo bonding. The layer displays good ECL activity and is stable over a long period of time. The excellent potential of this system for ECL sensing applications is demonstrated using the well-known ECL coreactant 2-(dibutylamino)ethanol (DBAE) as a model analyte, which can be detected to a level of 10 nM with a linear range between 10(-8) and 10(-4) M.

Simultaneous control of spectroscopic and electrochemical properties in functionalised electrochemiluminescent tris(2,2'-bipyridine)ruthenium(II) complexes.

Using a combination of electrochemical, spectroscopic and computational techniques, we have explored the fundamental properties of a series of ruthenium diimine complexes designed for coupling with other molecules or surfaces for electrochemiluminescence (ECL) sensing applications. With appropriate choice of ligand functionality, it is possible to manipulate emission wavelengths while keeping the redox ability of the complex relatively constant. DFT calculations show that in the case of electron withdrawing substituents such as ester or amide, the excited state is located on the substituted bipyridine ligand whereas in the case of alkyl functionality it is localised on a bipyridine. The factors that dictate annihilation ECL efficiency are interrelated. For example, the same factors that determine ΔG for the annihilation reaction (i.e. the relative energies of the HOMO and LUMO) have a corresponding effect on the energy of the excited state product. As a result, most of the complexes populate the excited state with an efficiency (Φ(ex)) of close to 80% despite the relatively wide range of emission maxima. The quantum yield of emission (Φ(p)) and the possibility of competing side reactions are found to be the main determinants of ECL intensity.

Electrochemiluminescent peptide nucleic acid-like monomers containing Ru(II)-dipyridoquinoxaline and Ru(II)-dipyridophenazine complexes.

A series of Ru(II)-peptide nucleic acid (PNA)-like monomers, [Ru(bpy)(2)(dpq-L-PNA-OH)](2+) (M1), [Ru(phen)(2)(dpq-L-PNA-OH)](2+) (M2), [Ru(bpy)(2)(dppz-L-PNA-OH)](2+) (M3), and [Ru(phen)(2)(dppz-L-PNA-OH)](2+) (M4) (bpy = 2,2'-bipyridine, phen = 1,10-phenanthroline, dpq-L-PNA-OH = 2-(N-(2-(((9H-fluoren-9-yl)methoxy)carbonylamino)ethyl)-6-(dipyrido[3,2-a:2',3'-c]phenazine-11-carboxamido)hexanamido)acetic acid, dppz-L-PNA-OH = 2-(N-(2-(((9H-fluoren-9-yl) methoxy)carbonylamino)ethyl)-6-(dipyrido[3,2-f:2',3'-h]quinoxaline-2-carboxamido)acetic acid) have been synthesized and characterized by IR and (1)H NMR spectroscopy, mass spectrometry, and elemental analysis. As is typical for Ru(II)-tris(diimine) complexes, acetonitrile solutions of these complexes (M1-M4) show MLCT transitions in the 443-455 nm region and emission maxima at 618, 613, 658, and 660 nm, respectively, upon photoexcitation at 450 nm. Changes in the ligand environment around the Ru(II) center are reflected in the luminescence and electrochemical response obtained from these monomers. The emission intensity and quantum yield for M1 and M2 were found to be higher than for M3 and M4. Electrochemical studies in acetonitrile show the Ru(II)-PNA monomers to undergo a one-electron redox process associated with Ru(II) to Ru(III) oxidation. A positive shift was observed in the reversible redox potentials for M1-M4 (962, 951, 936, and 938 mV, respectively, vs Fc(0/+) (Fc = ferrocene)) in comparison with [Ru(bpy)(3)](2+) (888 mV vs Fc(0/+)). The ability of the Ru(II)-PNA monomers to generate electrochemiluminescence (ECL) was assessed in acetonitrile solutions containing tripropylamine (TPA) as a coreactant. Intense ECL signals were observed with emission maxima for M1-M4 at 622, 616, 673, and 675 nm, respectively. At an applied potential sufficiently positive to oxidize the ruthenium center, the integrated intensity for ECL from the PNA monomers was found to vary in the order M1 (62%) > M3 (60%) > M4 (46%) > M2 (44%) with respect to [Ru(bpy)(3)](2+) (100%). These findings indicate that such Ru(II)-PNA bioconjugates could be investigated as multimodal labels for biosensing applications.

A Comparison of Commercially Available Screen-Printed Electrodes for Electrogenerated Chemiluminescence Applications.

We examined a series of commercially available screen-printed electrodes (SPEs) for their suitability for electrochemical and electrogenerated chemiluminescence (ECL) detection systems. Using cyclic voltammetry with both a homogeneous solution-based and a heterogeneous bead-based ECL assay format, the most intense ECL signals were observed from unmodified carbon-based SPEs. Three commercially available varieties were tested, with Zensor outperforming DropSens and Kanichi in terms of sensitivity. The incorporation of nanomaterials in the electrode did not significantly enhance the ECL intensity under the conditions used in this evaluation (such as gold nanoparticles 19%, carbon nanotubes 45%, carbon nanofibers 21%, graphene 48%, and ordered mesoporous carbon 21% compared to the ECL intensity of unmodified Zensor carbon electrode). Platinum and gold SPEs exhibited poor relative ECL intensities (16% and 10%) when compared to carbonaceous materials, due to their high rates of surface oxide formation and inefficient oxidation of tri-n-propylamine (TPrA). However, the ECL signal at platinum electrodes can be increased ∼3-fold with the addition of a surfactant, which enhanced TPrA oxidation due to increasing the hydrophobicity of the electrode surface. Our results also demonstrate that each SPE should only be used once, as we observed a significant change in ECL intensity over repeated CV scans and SPEs cannot be mechanically polished to refresh the electrode surface.

Tuning the electrochemiluminescent properties of iridium complexes of N-heterocyclic carbene ligands.

A series of five heteroleptic Ir(iii) complexes of the general form Ir(dfppy)2(C^C) have been prepared (where dfppy represents 2-(2,4-difluorophenyl)pyridine and C^C represents a bidentate cyclometalated phenyl substituted imidazolylidene ligand). The cyclometalated phenyl ring of the imidazolylidene ligand was either unsubstituted or substituted with electron donating (OMe and Me) or electron withdrawing (Cl and F) groups in the 2 and 4 positions. The synthesised Ir(iii) complexes have been characterised by elemental analysis, NMR spectroscopy, cyclic voltammetry and electronic absorption and emission spectroscopy. The molecular structures for four Ir(iii) complexes were determined by single crystal X-ray diffraction. Each of the Ir(iii) complexes exhibited intense photoluminescence in acetonitrile solution at room temperature with quantum yields (ΦPL) ranging from 58% to 86%. Cyclic voltammetry experiments revealed one oxidation process (formally ascribed to the metal centre), and two ligand-based reductions for each complex. Complexes 1-5 gave moderate to intense annihilation and co-reactant electrochemiluminescence (ECL). Consideration of the electrochemical, spectroscopic and theoretical investigations provide insights into the electrochemiluminescence behaviour.

A comparison of novel organoiridium(III) complexes and their ligands as a potential treatment for prostate cancer.

A range of 1,4-substituted 2-pyridyl-N-phenyl triazoles were synthesised and evaluated for their antiproliferative properties against lymph node cancer of the prostate (LNCaP) and bone metastasis of prostate cancer (PC-3) cells. Excellent-to-low IC50 values were determined (5.6-250 µM), and a representative group of 4 ligands were then complexed to iridium(III) giving highly luminescent species. Re-evaluation of these compounds against both cell lines was then undertaken and improved potency (up to 72-fold) was observed, giving IC50 values of 0.36-11 µM for LNCaP and 0.85-5.9 µM for PC-3. Preliminary screens for in vivo toxicity were conducted using a zebrafish model showing a wide range of induced toxicity depending of the compound evaluated. Apoptosis and Caspase-3 levels were also determined and showed no statistical difference between some of the treated specimens and the controls. This study may identify novel therapeutic agents for advanced stage of prostate cancer in humans.

New perspectives on the annihilation electrogenerated chemiluminescence of mixed metal complexes in solution.

Preliminary explorations of the annihilation electrogenerated chemiluminescence (ECL) of mixed metal complexes have revealed opportunities to enhance emission intensities and control the relative intensities from multiple luminophores through the applied potentials. However, the mechanisms of these systems are only poorly understood. Herein, we present a comprehensive characterisation of the annihilation ECL of mixtures of tris(2,2'-bipyridine)ruthenium(ii) hexafluorophosphate ([Ru(bpy)3](PF6)2) and fac-tris(2-phenylpyridine)iridium(iii) ([Ir(ppy)3]). This includes a detailed investigation of the change in emission intensity from each luminophore as a function of both the applied electrochemical potentials and the relative concentrations of the two complexes, and a direct comparison with two mixed (Ru/Ir) ECL systems for which emission from only the ruthenium-complex was previously reported. Concomitant emission from both luminophores was observed in all three systems, but only when: (1) the applied potentials were sufficient to generate the intermediates required to form the electronically excited state of both complexes; and (2) the concentration of the iridium complex (relative to the ruthenium complex) was sufficient to overcome quenching processes. Both enhancement and quenching of the ECL of the ruthenium complex was observed, depending on the experimental conditions. The observations were rationalised through several complementary mechanisms, including resonance energy transfer and various energetically favourable electron-transfer pathways.

Potential-Resolved Electrogenerated Chemiluminescence for the Selective Detection of Multiple Luminophores.

Electrogenerated chemiluminescence (ECL) is fundamentally dependent on the applied electrode potential, and measuring ECL intensity over a range of different potentials is commonly used to examine the underlying chemical reaction pathways responsible for the emission of light. Several research groups have now demonstrated that the applied potential can be exploited to selectively elicit ECL from: 1) multiple excited states within a single chemical species; 2) multiple emitters sharing a common co-reactant; or 3) distinct ECL systems. This new generation of multiplexed ECL processes has been facilitated by the extensive development of novel electrochemiluminophores and instrumental approaches such as the near-continuous collection of ECL spectra with CCD detectors during voltammetry or chronoamperometry experiments.

Frontispiece: Potential-Resolved Electrogenerated Chemiluminescence for the Selective Detection of Multiple Luminophores.

The frontispiece shows a representative graph of a mixed electrogenerated chemiluminescence system, resolved by both the potential applied to initiate the reactions and the distinct spectral distribution of the three emitters. The Review by Paul S. Francis et al. on page 456, outlines recent innovation in the area of potential-resolved ECL, which can be exploited to selectively elicit light from multiple excited states within a single chemical species, multiple emitters sharing a common co-reactant, or distinct ECL systems within the same solution.