Determination of Dansyl Amino Acids and Oxalate by HPLC with Electrogenerated Chemiluminescence Detection Using Tris(2,2'-bipyridyl)ruthenium(II) in the Mobile Phase

A new electrogenerated chemiluminescence detection method is investigated for use in detection in reversed-phase and reversed-phase ion-pair HPLC with Ru(bpy)(3)(2+) in the mobile phase. In this method, different concentrations of Ru(bpy)(3)(2+) are dissolved in the mobile phase and the HPLC column flushed with the mobile phase for 1 h until the column is saturated with Ru(bpy)(3)(2+). The separated analytes along with Ru(bpy)(3)(2+) pass through an optical-electrochemical flow cell which has a dual platinum electrode held at a potential of 1250 mV vs a Ag/AgCl reference electrode. On the surface of the electrode, Ru(bpy)(3)(2+) is oxidized to Ru(bpy)(3)(3+) which reacts with the analytes to emit light. The retention times, retention orders, detection limits, and linearity in working curves are compared to those obtained with the conventional postcolumn Ru(bpy)(3)(2+) addition method. The retention times for dansyl amino acids with Ru(bpy)(3)(2+) in the mobile phase are longer than those obtained with the postcolumn addition approach. This may be caused by π-to-π interactions between the aromatic groups of the dansyl derivatives and the bipyridyl groups of Ru(bpy)(3)(2+) in the Ru(bpy)(3)(2+)-saturated reversed-phase column. Similarly, oxalate is separated from urine and blood plasma samples by reversed-phase ion-pair HPLC. Plasma samples are obtained using ultrafiltration to remove proteins from whole blood. Retention times for oxalate with the two detection techniques are identical, and detection limits for these techniques are compared.     

Electrogenerated chemiluminescence from R(bpy)3(2+) ion-exchanged in carbon nanotube/perfluorosulfonated ionomer composite films

The electrochemistry and electrogenerated chemiluminescence (ECL) of ruthenium(II) tris(bipyridine) (Ru(bpy)(3)(2+)) ion-exchanged in carbon nanotube (CNT)/Nafion composite films were investigated with tripropylamine (TPA) as a coreactant at a glassy carbon (GC) electrode. The major goal of this work was to investigate and develop new materials and immobilization approaches for the fabrication of ECL-based sensors with improved sensitivity, reactivity, and long-term stability. Ru(bpy)(3)(2+) could be strongly incorporated into Nafion film, but the rate of charge transfer was relative slow and its stability was also problematic. The interfusion of CNT in Nafion resulted in a high peak current of Ru(bpy)(3)(2+) and high ECL intensity. The results indicated that the composite film had more open structures and a larger surface area allowing faster diffusion of Ru(bpy)(3)(2+) and that the CNT could adsorb Ru(bpy)(3)(2+) and also acted as conducting pathways to connect Ru(bpy)(3)(2+) sites to the electrode. In the present work, the sensitivity of the ECL system at the CNT/Nafion film-modified electrodes was more than 2 orders of magnitude higher than that observed at a silica/Nafion composite film-modified electrode and 3 orders of magnitude higher than that at pure Nafion films. The CNT/Nafion composite film-modified GC electrodes also exhibited long-term stability.     

Electrochemiluminescence of Tris(2,2'-bipyridine)ruthenium in Water at Carbon Microelectrodes

Electrochemiluminescence (ECL) of Ru(bpy)(3)(2+) in water only, without any added electrolyte or reducing agents, has been obtained at carbon interdigitated microelectrode arrays (C-IDAs) of 2 μm width and spacing. In a generation/collection biasing mode, ECL can be clearly seen with the naked eye in normal room lighting at concentrations greater than 1 mM. Using a conventional photomultiplier tube (PMT), a detection limit of 10(-)(7) M Ru(bpy)(3)(2+) has been achieved for an electrode area of 0.25 mm(2). In comparison, the ECL intensity produced at Pt-IDA of the same geometry, under identical experimental conditions, was more than 300 times less. The ECL obtained at C-IDAs is attributed to the annihilation reaction of the reduced and oxidized forms of the Ru(bpy)(3)(2+) made possible due to the small electrode spacing.     

Multilabeling biomolecules at a single site. 1. Synthesis and characterization of a dendritic label for electrochemiluminescence assays

Ultrasensitive bioanalytical assays are of great value for early detection of human diseases and pathogens. The sensitivities of immunoassays and DNA probing can be enhanced by multilabeling the biorecognition partner used for affinity-based assays. However, the bioreactivity of biomolecules is affected by a high degree of multilabeling at multiple functional sites. It is proposed that dendritic scaffoldings be used to link multiple signal-generating units to a single site with potentially minimum impact on the bioaffinity. A prototype label, a zeroth-generation dendron, bearing three [Ru(bpy)(3)](2+) units for electrochemiluminescence (ECL) assays was synthesized and characterized preliminarily by spectroscopic, electrochemical, and ECL methods. No evidence of interaction between the neighboring [Ru(bpy)(3)](2+) units in the label molecule was found from these characterizations. Both the photoluminescence and ECL of the prototype label have features very similar to those of mononuclear [Ru(bpy)(3)](2+) compounds. Labeling a model protein with a triad of [Ru(bpy)(3)](2+) at one NH(2) position was demonstrated. The results reported here provide support to applying the proposed multilabeling strategy to affinity-based bioanalytical assays.     

Method for effective immobilization of Ru(bpy)(3)2+ on an electrode surface for solid-state electrochemiluminescene detection

A novel method for effective immobilization of Ru(bpy)3(2+) on an electrode surface is developed. The whole process involves two steps: the electrostatic interactions between citrate-capped gold nanoparticles (AuNPs) and Ru(bpy)3Cl2 in aqueous medium were used to fabricate Ru(bpy)(3)2+-AuNP aggregates (Ru-AuNPs) first, and then the Au-S interactions between as-formed Ru-AuNPs and sulfhydryl groups were used to effectively immobilize the Ru-AuNPs on a sulfhydryl-derivated indium tin oxide (ITO) electrode surface. As-prepared ITO electrode shows excellent stability, and the ECL active species Ru(bpy)3(2+) contained therein exhibit excellent ECL behaviors.     

Electrogenerated chemiluminescence. 72. Determination of immobilized DNA and C-reactive protein on Au(111) electrodes using tris(2,2'-bipyridyl)ruthenium(II) labels

Anodic electrogenerated chemiluminescence (ECL) with tri-n-propylamine (TPrA) as a coreactant was used to determine DNA and C-reactive protein (CRP) by immobilizations on Au(111) electrodes using tris(2,2'-bipyridyl)ruthenium(II) (Ru(bpy)(3)(2+)) labels. A 23-mer synthetic single-stranded (ss) DNA derived from the Bacillus anthracis with an amino-modified group at the 5' end position was covalently attached to the Au(111) substrate precoated with a self-assembled thiol monolayer of 3-mercaptopropanoic acid (3-MPA) in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) and then hybridized with a target ssDNA tagged with Ru(bpy)(3)(2+) ECL labels. Similarly, biotinylated anti-CRP species were immobilized effectively onto the Au(111) substrate precovered with a layer of avidin linked covalently via the reaction between avidin and a mixed thiol monolayer of 3-MPA and 16-mercaptohexadecanoic acid on Au(111) in the presence of EDAC and N-hydroxysuccinimide. CRP and anti-CRP tagged with Ru(bpy)(3)(2+) labels were then conjugated to the surface layer. ECL responses were generated from the modified electrodes described above by immersing them in a TPrA-containing electrolyte solution. A series of electrode treatments, including blocking free -COOH groups with ethanol amine, pinhole blocking with bovine serum albumin, washing with EDTA/NaCl/Tris buffer, and spraying with inert gases, were used to reduce the nonspecific adsorption of the labeled species. The ECL peak intensity was linearly proportional to the analyte CRP concentration over the range 1-24 microg/mL. CRP concentrations of two unknown human plasma/serum specimens were measured by the standard addition method based on this technique.     

Spectroelectrochemical sensing based on multimode selectivity simultaneously achievable in a single device. 2. Demonstration of selectivity in the presence of direct interferences

Three modes of selectivity based on charge-selective partitioning, electrolysis potential, and spectral absorption wavelength were demonstrated simultaneously in a new type of spectroelectrochemical sensor. Operation and performance of the three modes of selectivity for detection of analytes in the presence of direct interferences were investigated using binary mixture systems. These binary mixtures consisted of Fe(CN)(6)(3-) and Ru(bpy)(3)(2+) and of Fe(CN)(6)(4-) and Ru(CN)(6)(4)(-) in aqueous solutions. Results on the Fe(CN)(6)(3-)/Ru(bpy)(3)(2+) binary mixture showed that an anion-exchange coating consisting of PDMDAAC-SiO(2) [where PDMDAAC is poly(dimethyldiallylammonium chloride)] and a cation-exchange coating consisting of Nafion-SiO(2) can trap and preconcentrate analytes with charge selection. At the same time, such coatings exclude interferences carrying the same type of charge as that of the exchange sites in the sensor coating. Using the Fe(CN)(6)(4-)/Ru(CN)(6)(4-) binary mixture, the Fe(CN)(6)(4-) component can be selectively detected by restricting the modulation potential cycled to a range specific to the redox-active Fe(CN)(6)(4-) component and simultaneously monitoring the optical response at the overlapping wavelength of 420 nm. It was also shown that, when the wavelength for optical monitoring was chosen as 500 nm, which is specific to the Ru(CN)(6)(4-) component, interference from the Fe(CN)(6)(4-) component for spectroelectrochemical detection of Ru(CN)(6)(4-) was significantly suppressed, even though the cyclic modulation potential encompassed the redox range for the Fe(CN)(6)(4-) component.     

Spectroelectrochemical sensing based on multimode selectivity simultaneously achievable in a single device. 16. Sensing by fluorescence

A fluorescence spectroelectrochemical sensor capable of detecting very low concentrations of metal complexes is described. The sensor is based on a novel spectroelectrochemical sensor that incorporates multiple internal reflection spectroscopy at an optically transparent electrode (OTE) coated with a selective film to enhance detection limits by preconcentrating the analyte at the OTE surface. Nafion was used as the selective cation exchange film for detecting Ru(bpy)(3)(2+), the model analyte, which fluoresces at 605 nm when excited with a 441.6-nm HeCd laser. The unoptimized linear dynamic range of the sensor for Ru(bpy)(3)(2+) is between 1 x 10(-)(11) and 1 x 10(-)(7) M with a calculated 2 x 10(-)(13) M detection limit. The sensor employs extremely thin films ( approximately 12 nm) without significantly sacrificing its sensitivity. The sensor response is demonstrated with varying film thicknesses. A state-of-the-art flow cell design allows variable cell volumes as low as approximately 4 microL. Fluorescence of the sample can be controlled by electromodulation between 0.7 and 1.3 V. Sensor operation is not reversible for the chosen model film (Nafion) and sample (Ru(bpy)(3)(2+)) but it can be regenerated with ethanol for multiple uses.     

Electrogenerated chemiluminescence sensors using Ru(bpy)3(2+) doped in silica nanoparticles

A novel electrogenerated chemiluminescence (ECL) sensor based on Ru(bpy)3(2+)-doped silica (RuDS) nanoparticles conjugated with a biopolymer chitosan membrane was developed. These uniform RuDS nanoparticles (approximately 40 nm) were prepared by a water-in-oil microemulsion method and were characterized by electrochemical and transmission electron microscopy technology. The Ru(bpy)3(2+)-doped interior maintained its high ECL efficiency, while the exterior nanosilica prevented the luminophor from leaching out into the aqueous solution due to the electrostatic interaction. This is the first attempt to branch out the application of RuDS nanoparticles into the field of ECL, and since a large amount of Ru(bpy)3(2+) was immobilized three-dimensionally on the electrode, the Ru(bpy)3(2+) ECL signal could be enhanced greatly, which finally resulted in the increased sensitivity. This sensor shows a detection limit of 2.8 nM for tripropylamine, which is 3 orders of magnitude lower than that observed at a Nafion-based ECL sensor. Furthermore, the present ECL sensor displays outstanding long-term stability.     

Spatial scanning spectroelectrochemistry. Study of the electrodeposition of Pd nanoparticles at the liquid/liquid interface

Spatial scanning spectroelectrochemistry is a new analytical technique that provides spectral information at different distances from an electrified liquid/liquid interface where an electrochemical process takes place. As a proof of concept, we have studied two different electrochemical processes at the electrified liquid/liquid interface: (1) Ru(bpy)(3)(2+) transfer through the water/1,2-dichloroethane interface and (2) electrodeposition of Pd nanoparticles at the water/1,2-dichloroethane interface. The instrumental setup developed consists of a movable slit for the light beam to sample at well-defined positions on both sides of the interface, providing important information about the chemical process occurring. If the slit is scanned at different distances from the interface during an electrochemical experiment, a complete picture of the reactions and equilibria in the diffusion layer can be obtained. For example, in the case of the Ru(bpy)(3)(2+), the experiments show clearly how the complex is transferred from one phase to the other. In the case of electrosynthesis of Pd nanoparticles, it is demonstrated that nanoparticles are not only deposited at the interface but diffuse to the aqueous bulk solution. These in situ observations were confirmed by ex situ experiments using transmission electron microscopy.     

Microchip device with 64-site electrode array for multiplexed immunoassay of cell surface antigens based on electrochemiluminescence resonance energy transfer

This paper describes a novel on-chip microarray platform based on an electrochemiluminescence resonance energy transfer (ECL-RET) strategy for rapid assay of cancer cell surface biomarkers. This platform consists of 64 antigen-decorated CdS nanorod spots with the diameter of 1.0 cm uniformly distributed on 16 indium tin oxide (ITO) strips, which is coated with a multichannel decorated polydimethylsiloxane (PDMS) slice to realize multiplexed determination of antigens. To shorten the immune reaction time in the microchannels and simplify the device, magnetic stirring and four-channel universal serial bus (USB) ports for plug-and-play were used. When Ru(bpy)(3)(2+) labeled antibodies were selectively captured by the corresponding antigens on the CdS nanorod spot array, ECL-RET from the CdS nanorod (donor) by cathodic emission in the presence of K(2)S(2)O(8) to Ru(bpy)(3)(2+) (acceptor) occurred. With signal amplification of Ru(bpy)(3)(2+) and competitive immunoassay, carcinoembryonic antigen (CEA), α-fetoprotein (AFP), and prostate specific antigen (PSA) as models were detected on this microfluidic device via recording the increased ECL-RET signals on electrode surfaces. Furthermore, this multiplexed competitive immunoassay was successfully used for detecting cancer cell surface antigens via the specific antibody-cell interactions and cell counting via cell surface receptors and antigens on the CdS nanorod surface. This platform provides a rapid and simple but sensitive approach with microliter-level sample volume and holds great promise for multiplexed detection of antigens and antigen-specific cells.     

Quenching of electrogenerated chemiluminescence by phenols, hydroquinones, catechols, and benzoquinones

Efficient quenching of Ru(bpy)(3)(2+) (bpy = 2,2'-bipyridine) electrogenerated chemiluminescence has been observed in the presence of phenols, catechols, hydroquinones, and benzoquinones. In most instances, quenching is observed with 100-fold excess of quencher over Ru(bpy)(3)(2+), with complete quenching observed between 1000- and 2000-fold excess. The mechanism of quenching is believed to involve energy transfer from the excited-state luminophore to benzoquinone. In the case of phenols, catechols, and hydroquinones, quenching is believed to occur via a benzoquinone derivative formed at the electrode surface. Photoluminescence and UV-visible experiments coupled with bulk electrolysis support the formation of benzoquinone products upon electrochemical oxidation.     

A single calibration graph for the direct determination of ascorbic and dehydroascorbic acids by electrogenerated luminescence based on Ru(bpy)(3)2+ in aqueous solution

Ascorbic (H2A) and dehydroascorbic (DA) acids were for the first time directly determined in a single chromatographic run by means of the tris(2,2'-bipyridine)ruthenium(II) (Ru(bpy)(3)2+) based electrogenerated chemiluminescence (ECL) detection. For the first time, it was demonstrated that DA, a nonelectroactive compound, is ECL active and is responsible for the ECL behavior of H2A. This fact, together with the lack of a DA standard, suggested the use of a calibration graph obtained for H2A, for determining both analytes. The proven ECL activity of DA, together with literature data relative to the standard redox potentials of the different species coming from H2A, led to a reconsideration of the proposed ECL reaction mechanism for H2A. The role of the OH- ion in the reaction mechanism of the two analytes appeared to be crucial. H2A and DA could be separated by a suitable C18-reversed-phase HPLC column using an aqueous 30 mM H3PO4 solution as the mobile phase. The optimal ECL response was achieved by polarizing the working electrode at 1.150 Vvs SCE (standard calomel electrode) (oxidation diffusion limiting potential for both H2A and Ru(bpy)(3)2+). The Ru(bpy)(3)2+ solution, at pH 10 for carbonate buffer, was mixed to the eluent solution in a postcolumn system, obtaining, still at pH 10, the final 0.25 mM Ru(bpy)(3)2+ concentration. The detection limit found for the two analytes was 1 x 10(-7) M. The method was successfully applied to the determination of the analytes in a commercially available orange fruit juice.     

The effects of nonionic surfactants on the tris(2,2'-bipyridyl)ruthenium(II)--tripropylamine electrochemiluminescence system

The electrochemistry and electrogenerated chemiluminescence (ECL) of Ru(bpy)3(2+) (bpy = 2,2'-bipyridyl) were studied in the presence of the nonionic surfactants Triton X-100, Thesit, and Nonidet P40. The anodic oxidation of Ru(bpy)3(2+) produces ECL in the presence of tri-n-propylamine in both aqueous and surfactant solutions. Increases in both ECL efficiency (> or =8-fold) and duration of the ECL signal were observed in surfactant media. A shift to lower energies of the Ru(bpy)3(2+) ECL emission by approximately 8 nm was also observed. The one-electron oxidation of Ru(bpy)3(2+) to Ru(bpy)3(3t) occurs at + 1.03 V vs Ag/AgCl in aqueous buffered (0.2 M potassium phosphate) solution as found by square wave voltammetry. This potential did not shift in surfactant systems, indicating that the redshifts in ECL emission are due to stabilization of ligand pi* orbitals in the metal-to-ligand charge-transfer excited state. These results are consistent with hydrophobic interactions between Ru(bpy)3(2+) and the nonionic surfactants.     

Chemiluminescence detection using regenerable tris(2,2'-bipyridyl)ruthenium(II) immobilized in Nafion.

The development of a detection method based on the electrogenerated chemiluminescence of tris(2,2'-bipyridine)ruthenium(II), (Ru(bpy)3(2+], immobilized in a Nafion film coated on an electrode is discussed. Control of the electrode potential controls creation of the reactive reagent Ru(bpy)3(3+) which reacts with certain analytes to yield chemiluminescence emission of intensity proportional to the analyte concentration. The reaction results in Ru(bpy)3(3+) being converted to Ru(bpy)3(2+), which then is recycled to Ru(bpy)3(3+) again at the electrode. This sensor has been used in flow injection to determine oxalate, alkylamines, and NADH. Detection limits are 1 microM, 10 nM, and 1 microM, respectively, with working ranges extending over 4 decades in concentration. Sensitivity is constant over the wide pH range from 3 to 10. With oxalate, and to a small extent with amines, emission intensities increase with increasing ionic strength; this was shown to be a phenomenon related to the Nafion film and not to the chemiluminescence reaction. Emission intensities increase with temperature. The sensor remains stable for several days with suitable storage conditions. Significant amounts of Ru(bpy)3(3+) are shown to be capable of storage within the film.     

Enhanced electrogenerated chemiluminescence in the presence of fluorinated alcohols

The electrochemistry, UV-vis absorption, photoluminescence (PL), and coreactant electrogenerated chemiluminescence (ECL) of Ru(bpy)3(2+) (where bpy=2,2'-bipyridine) have been obtained in a series of hydroxylic solvents. The solvents included fluorinated and nonfluorinated alcohols and alcohol/water mixtures. Tri-n-propylamine was used as the oxidative-reductive ECL coreactant. Blue shifts of up to 30 nm in PL emission wavelength maximums are observed compared to a Ru(bpy)3(2+)/H2O standard due to interactions of the polar excited state (i.e., *Ru(bpy)3(2+)) with the solvent media. For example, Ru(bpy)3(2+) in water has an emission maximum of 599 nm while in the more polar hexafluoropropanol and trifluoroethanol it is 562 and 571 nm, respectively. ECL spectra are similar to PL spectra, indicating the same excited state is formed in both experiments. The difference between the electrochemically reversible oxidation (Ru(bpy)3(2+/3+)) and first reduction (Ru(bpy)2(2+/1+)) correlates well with the energy gap observed in the luminescence experiments. Although the ECL is linear in all solvents with [Ru(bpy)3(2+)] ranging from 100 to 0.1 nm, little correlation between the polarity of the solvent and the ECL efficiency (phiecl=number of photons per redox event) was observed. However, dramatic increases in phiecl ranging from 6- to 270-fold were seen in mixed alcohol/water solutions.     

Scanning electrochemical microscopy. 32. Gallium ultramicroelectrodes and their application in ion-selective probes

Gallium ultramicroelectrodes for amperometric measurements in scanning electrochemical microscopy were fabricated by introduction of liquid Ga into drawn glass micropipets. Cyclic voltammetry of Ru(NH(3))(6)(3+) and the use of this species for SECM imaging is described. Double-barrel micropipet tips with a Ga amperometric electrode and an ion-selective (K(+)) potentiometric probe can also be constructed. This probe was used to image the K(+) activity near a 20-μm-diameter lumen of a glass capillary.     

Analysis of the photodecomposition products of Ru(bpy)3(2+) in various buffers and upon zeolite encapsulation

The photodecomposition products of Ru(bpy)3(2+) in water, in aqueous buffered solutions and encapsulated in zeolite-Y have been analyzed by chromatography and UV-visible spectroscopy. The chromatographic method is found to be capable of separating species with the same charge but slightly different ligands as well as geometrical isomers. In all the systems investigated, photodecomposition proceeded via photoaquation resulting in the formation of cis- and trans-Ru(bpy)2(OH2)2(2+). In the case of acetate and phthalate buffers, a third species, Ru(bpy)2(L)(OH2)+, where L is the buffer anion, was found to be the dominant product. For a given pH, the extent of decomposition was found to be dependent on both the buffer anion, following the trend, phosphate < acetate < phthalate and buffer concentration. The presence of the electron-transfer quenching agent, N,N'-dimethyl-4,4'-bipyridinium ion in the medium led to a decrease of the photodecomposition and closely followed the quenching efficiency as measured by intensity and lifetime quenching studies. Encapsulation of Ru(bpy)3(2+) in the supercages of zeolite-Y did not lead to a substantial decrease in photodecomposition as compared to an aqueous solution, suggesting that the expected enhanced stability of Ru(bpy)3(2+*) due to the destabilization of 3dd orbitals and the cage effect was being negated by the close proximity and intrazeolite packing of H2O molecules around the Ru center.     

Imaging size-selective permeation through micropatterned thin films using scanning electrochemical microscopy

This paper describes a new approach for quantification of rates of molecular transport through patterned, or otherwise heterogeneous, porous films supported on conductive substrates. Scanning Electrochemical Microscopy (SECM) has been used to image molecular sieving of redox active probes by thin, electropolymerized films of Fe(5-amino-1,10-phenanthroline)3(2+) on micropatterned and microdisk array electrodes. Films as thin as 12 nm completely block redox mediators with average molecular diameters greater than 12 A, whereas smaller diameter probes (radii 5-8 A) were observed to permeate selectively. SECM tip currents measured for three different redox permeants/mediators are observed to decrease with increasing polymer thickness, consistent with a transport model that includes partitioning into and diffusion within the polymer films. Permeabilities, PDf, within the poly[Fe(5-NH2-phen)3(2+)] films have been quantitatively determined from the SECM tip currents and are in excellent agreement with data previously obtained from rotatingdisk electrochemistry. This new methodology provides a versatile approach for quantitative investigation of membrane transport and permeation selectivity with good lateral spatial resolution.     

Electrogenerated chemiluminescence from tris(2,2'-bipyridyl)ruthenium(II) immobilized in titania-perfluorosulfonated ionomer composite films

Electrochemical behavior and electrogenerated chemiluminescence (ECL) of tris(2,2'-bipyridyl)ruthenium(II) (Ru(bpy)3(2+)) immobilized in sol-gel-derived titania TiO2)-Nafion composite films coated on a glassy carbon electrode have been investigated. The electroactivity of Ru(bpy)3(2+) ion exchanged into the composite films and its ECL behavior were strongly dependent upon the amount of Nafion incorporated into the TiO2-Nafion composite films. The ECL sensor of Ru(bpy)32+ immobilized in a TiO2-Nafion composite with 50% Nafion content showed the maximum ECL intensities for both tripropylamine (TPA) and sodium oxalate in 0.05 M phosphate buffer solution at pH 7. Detection limits were 0.1 microM for TPA and 1.0 microM for oxalate (S/N = 3) with a linear range of 3 orders of magnitude in concentration. The present ECL sensor showed improved ECL sensitivity and long-term stability, as compared to the ECL sensors based on pure Nafion films. The present Ru(bpy)3(2+) ECL sensor based on TiO2-Nafion (50%) composite films was applied as an HPLC detector for the determination of erythromycin in human urine samples. The present Ru(bpy)3(2+) ECL sensor was stable in the mobile phase containing a high content of organic solvent (30%, v/v), in contrast to a pure Nafion-based Ru(bpy)3(2+) ECL sensor. The detection limit for erythromycin was 1.0 microM, with a linear range of 3 orders of magnitude in concentration.