Direct detection of DNA with an electrocatalytic threading intercalator

Herein we report the synthesis, intercalating properties, and analytical applications of an imidazole-substituted naphthalene diimide, N,N'-bis(3-propylimidazole)-1,4,5,8-naphthalene diimide (PIND), functionalized with electrocatalytic redox moieties. PIND was prepared in a single-step reaction from the corresponding dianhydride. Attachment of the redox moieties to PIND relied upon ligand exchange with one of the liable chloride ligands of an Os(bpy)2Cl2 (bpy = 2,2'-bipyridine) complex. The Os(bpy)2Cl2 complex was grafted onto PIND through coordinative bonds with the two imidazole groups at its termini, forming a PIND-[Os(bpy)2Cl]+ compound (PIND-Os). Gel electrophoretic studies revealed that PIND-Os binds more strongly to double-stranded DNA (ds-DNA) than its parent compound 1,4,5,8-naphthalene diimide. The naphthalene diimide group binds to ds-DNA in a "classical" threading intercalation mode, while the two Os(bpy)2Cl+ pendants interact with DNA via electrostatic interaction, reinforcing the intercalation by "locking up" the naphthalene diimide group in place. An electrochemical biosensor was fabricated using the redox-active and catalytic PIND-Os intercalator. An increase in sensitivity of 2500-fold over direct voltammetry was obtained in electrocatalytic amperometry, making this an interesting system for amperometric DNA sensing. Under optimized experimental conditions, the biosensor allowed the detection of a 50-mer target DNA in the range of 1.0-300 pM with a detection limit of 600 fM (1.5 amol, 23 fg).     

A nucleic acid biosensor for gene expression analysis in nanograms of mRNA

An ultrasensitive nucleic acid biosensor for direct detection of genes in mRNA extracted from animal tissues is described. It is based on amperometric detection of a target gene by forming an mRNA/redox polymer bilayer on a gold electrode. The mRNA was directly labeled with cisplatin-biotin conjugates through coordinative bonds with purine bases in the mRNA molecules. A subsequent binding of glucose oxidase-avidin conjugates to the labeled mRNA and the introduction of a poly(vinylimidazole-co-acrylamide) partially imidazole-complexed with [Os(bpy)(2)(im)] (bpy = 2,2'-bipyridine, im = imidazole) redox polymer overcoating to the electrode allowed for electrochemical detection of the oxidation current of glucose in solution. Depending on individual genes, detection limits of subfemtograms were achieved. As compared to a sandwich-type assay, the sensitivity was improved by as much as 25-fold through the incorporation of multiple enzyme labels to the mRNA molecules. Less than 2-fold gene expression difference was unambiguously differentiated in as little as 5.0 ng of mRNA. With the greatly improved sensitivity, at least 1000-fold more sensitive than fluorescence-based techniques, the amount of mRNA needed in the assay was cut down from microgram to nanogram levels.     

Real-Time Electrochemical PCR with a DNA Intercalating Redox Probe

The proof-of-principle of a nonoptical real-time PCR method based on the electrochemical monitoring of a DNA intercalating redox probe that becomes considerably less easily electrochemically detectable once intercalated to the amplified double-stranded DNA is demonstrated. This has been made possible thanks to the finding of a redox intercalator that (i) strongly and specifically binds to the amplified double-stranded DNA, (ii) does not significantly inhibit PCR, (iii) is chemically stable under PCR cycling, and (iv) is sensitively detected by square wave voltammetry during PCR cycling. Among the different DNA intercalating redox probes that we have investigated, namely, methylene blue, Os[(bpy)(2)phen](2+), Os[(bpy)(2)DPPZ](2+), Os[(4,4'-dimethyl-bpy)(2)DPPZ](2+) and Os[(4,4'-diamino-bpy)(2)DPPZ](2+) (with bpy = 2,2'-bipyridine, phen = phenanthroline, and DPPZ = dipyrido[3,2-a:2',3'-c]phenazine), the one and only compound with which it has been possible to demonstrate the proof-of-concept is the Os[(bpy)(2)DPPZ](2+). In terms of analytical performances, the methodology described here compares well with optical-based real-time PCRs, offering finally the same advantages than the popular and routinely used SYBR Green-based real-time fluorescent PCR, but with the additional incomes of being potentially much cheaper and easier to integrate in a hand-held miniaturized device.     

Electron-transfer studies with a new flavin adenine dinucleotide dependent glucose dehydrogenase and osmium polymers of different redox potentials

A new extracellular flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase from Glomerella cingulata (GcGDH) was electrochemically studied as a recognition element in glucose biosensors. The redox enzyme was recombinantly produced in Pichia pastoris and homogeneously purified, and its glucose-oxidizing properties on spectrographic graphite electrodes were investigated. Six different Os polymers, the redox potentials of which ranged in a broad potential window between +15 and +489 mV versus the normal hydrogen electrode (NHE), were used to immobilize and "wire" GcGDH to the spectrographic graphite electrode's surface. The GcGDH/Os polymer modified electrodes were evaluated by chronoamperometry using flow injection analysis. The current response was investigated using a stepwisely increased applied potential. It was observed that the ratio of GcGDH/Os polymer and the overall loading of the enzyme electrode significantly affect the performance of the enzyme electrode for glucose oxidation. The best-suited Os polymer [Os(4,4'-dimethyl-2,2'-bipyridine)(2)(PVI)Cl](+) had a potential of +309 mV versus NHE, and the optimum GcGDH/Os polymer ratio was 1:2 yielding a maximum current density of 493 μA·cm(-2) at a 30 mM glucose concentration.     

Voltammetric sensor for oxidized DNA using ultrathin films of osmium and ruthenium metallopolymers

Films containing [Os(bpy)2(PVP)10Cl]+ and [Ru(bpy)2(PVP)10Cl]+ metallopolymers were assembled layer by layer on pyrolytic graphite electrodes to make sensors that selectively detect oxidized DNA. These films showed reversible, independent electrochemistry for electroactive Os3+/Os2+ and Ru3+/Ru2+ centers, with formal potentials of 0.34 and 0.76 V vs SCE, respectively. The combination of ruthenium and osmium metallopolymers in the films provided a catalytic Os square wave voltammetry (SWV) peak that is mainly selective for 8-oxoguanine and the detection of other oxidized nucleobases from the Ru peak. The method is applicable to measurements on DNA in solution or DNA incorporated into films. Using the Os SWV peak, 1 oxidized nucleobase in 6000 was detected. The sensor is simple and inexpensive, and the approach may be useful for the detection of oxidized DNA as a clinical biomarker for oxidative stress.     

Use of polymeric indicator for electrochemical DNA sensors: poly(4-vinylpyridine) derivative bearing [Os(5,6-dimethyl-1,10-phenanthroline)2Cl]2+

A poly(4-vinylpyridine) (PVP) derivative bearing redox-active osmium complexes, PVP-[Os(5,6-dmphen)(2)Cl](2+) (5,6-dmphen = 5,6-dimethyl-1,10-phenanthroline), was employed as a hybridization indicator for electrochemical DNA sensors. PVP-[Os(5,6-dmphen)(2)Cl](2+) exhibited approximately 1000 times higher sensitivity than the corresponding monomeric analogue, [Os(5,6-dmphen)(3)](2+), in DNA determination due to polymeric effects. The detection limit of the present sensor was approximately 0.5 amol. Another merit of the polymeric indicator is that the redox potential was found to be +360 mV (vs Ag/AgCl), which is significantly lower than that reported for the monomeric analogue (+672 mV). The polymeric indicator was applicable to the discrimination of single- and double-base-mismatched DNAs from fully matched target DNA. The polymeric indicator can be removed from the electrode surface by rinsing the electrode in a high-temperature buffer for 6 min, and thus, the polymeric indicator-based DNA sensor can be used repeatedly.     

DNA biosensor for detection of Helicobacter pylori using phen-dione as the electrochemically active ligand in osmium complexes

A surface-based method for the study of the interactions of DNA with redox-active 1,10-phenantroline-5,6-dione (phen-dione) osmium complexes is described. The study was carried out using gold electrodes modified with DNA via adsorption and [Os(bpy)(2)(phe-dione)](3+/2+) (bpy = 2,2'-bipyridyl) or [Os(phen)(2)(phen-dione)](3+/2+) (phen = 1,10-phenantroline) as electrochemical reported molecules. The method, which is simple and reagent-saving, allows the accumulation of osmium complexes within the DNA layer. The amount of osmium complex bound by the adsorbed layer of DNA was determined from the voltammetric charge associated with the osmium redox process of the immobilized metal complex. The quinone moiety of the phen-dione ligand was useful as an indicator for electrochemical DNA sensing because of its redox response at low potentials. A thiol-linked single-stranded Helicobacter pylori DNA probe was immobilized, through S-Au bonds on to a gold electrode (density of modification 86 pmol/cm(2)). Following hybridization with the complementary DNA sequence, the osmium complex was electrochemically accumulated within the double-stranded DNA layer. Electrochemical detection was performed by differential pulse voltammetry over the potential range where the quinone moiety was redox active (i.e., at very low potentials, -0.020 V vs SSCE); with this approach, a sequence of the H. pylori could be quantified over the range from 5 to 20 pmol with a linear correlation of r = 0.9888 and a detection limit of approximately 6 pmol.     

Amperometric biosensors based on redox polymer-carbon nanotube-enzyme composites

Based on their size and unique electrical properties, carbon nanotubes offer the exciting possibility of developing ultrasensitive, electrochemical biosensors. In this study, we describe the construction of amperometric biosensors based on the incorporation of single-walled carbon nanotubes modified with enzyme into redox polymer hydrogels. The composite films were constructed by first incubating an enzyme in a single-walled carbon nanotube (SWNTs) solution and then cross-linking within a poly[(vinylpyridine)Os(bipyridyl)(2)Cl(2+/3+)] polymer film. Incorporation of SWNTs, modified with glucose oxidase, into the redox polymer films resulted in a 2-10-fold increase in the oxidation and reduction peak currents during cyclic voltammetry, while the glucose electrooxidation current was increased 3-fold to approximately 1 mA/cm(2) for glucose sensors. Similar effects were also observed when SWNTs were modified with horseradish peroxidase prior to incorporation into redox hydrogels.     

Cross-linked redox gels containing glucose oxidase for amperometric biosensor applications

Oxidoreductases, such as glucose oxidase, can be electrically "wired" to electrodes by electrostatic complexing or by covalent binding of redox polymers so that the electrons flow from the enzyme, through the polymer, to the electrode. We describe two materials for amperometric biosensors based on a cross-linkable poly(vinylpyridine) complex of [Os-(bpy)2Cl]+2+ that communicates electrically with flavin adenine dinucleotide redox centers of enzymes such as glucose oxidase. The uncomplexed pyridines of the poly(vinylpyridine) are quaternized with two types of groups, one promoting hydrophilicity (2-bromoethanol or 3-bromopropionic acid), the other containing an active ester (N-hydroxysuccinimide) that forms amide bonds with both lysines on the enzyme surface and with an added polyamine cross-linking agent (triethylenetetraamine, trien). In the presence of glucose oxidase and trien this polymer forms rugged, cross-linked, electroactive films on the surface of electrodes, thereby eliminating the requirement for a membrane for containing the enzyme and redox couple. The glucose response time of the resulting electrodes is less than 10 s. The glucose response under N2 shows an apparent Michaelis constant, Km' = 7.3 mM, and limiting current densities, jmax, between 100 and 800 microA/cm2. Currents are decreased by 30-50% in air-saturated solutions because of competition between O2 and the Os(III) complex for electrons from the reduced enzyme. Rotating ring desk experiments in air-saturated solutions containing 10 mM glucose show that about 20% of the active enzyme is electrooxidized via the Os(III) complex, while the rest is oxidized by O2. These results suggest that only part of the active enzyme is in electrical contact with the electrode.     

Antioxidant redox sensors based on DNA modified carbon screen-printed electrodes

Antioxidant redox sensors based on DNA modified carbon screen-printed electrodes were developed. The carbon ink was doped with TiO2 nanoparticles, onto which double-strand DNA was adsorbed. A redox mediator, namely, tris-2,2'-bipyridine ruthenium(II) [Ru(bpy)3(2+)] was electrooxidized on the electrode surface to subsequently oxidize both the adsorbed ds-DNA and the antioxidants in solution. The resulting oxidation damage of the adsorbed ds-DNA was then detected by square wave voltammetry in a second solution containing only Ru(bpy)3Cl2 at a low concentration (microM). A kinetic model was developed to study the protecting role of antioxidants in aqueous solutions. The electrochemical sensor has been applied to evaluate the redox antioxidant capacity of different molecules.     

A lactate electrochemical biosensor with a titanate nanotube as direct electron transfer promoter

Hydrogen titanate (H(2)Ti(3)O(7)) nanotubes (TNTs) have been synthesized by a one-step hydrothermal processing. Lactate oxidase (LOx) enzyme has been immobilized on the three-dimensional porous TNT network to make an electrochemical biosensor for lactate detection. Cyclic voltammetry and amperometry tests reveal that the LOx enzyme, which is supported on TNTs, maintains their substrate-specific catalytic activity. The nanotubes offer the pathway for direct electron transfer between the electrode surface and the active redox centers of LOx, which enables the biosensor to operate at a low working potential and to avoid the influence of the presence of O(2) on the amperometric current response. The biosensor exhibits a sensitivity of 0.24?μA?cm(-2)?mM(-1), a 90% response time of 5?s, and a linear response in the range from 0.5 to 14?mM and the redox center of enzyme obviates the need of redox mediators for electrochemical enzymatic sensors, which is attractive for the development of reagentless biosensors.     

Detection of glucose at 2 fM concentration

We report the amperometric detection of glucose at 2 fM concentration in a physiological buffer solution at 1 atm O2 pressure. The sensitive assay is based on the close to absolute electroreductive stripping of O2 from the solution near the glucose electrooxidizing anode. The glucose was detected by its electrooxidation on a stationary glassy carbon disk surrounded by an also stationary platinum ring. The disk was coated with a film of glucose oxidase (GOx), electrically "wired" with PVP-[Os(N,N'-dimethyl-2,2'-biimidazole)3]2+/3+ (polymer I), having a redox potential of -0.19 V versus Ag/AgCl. The ring was coated with bilirubin oxidase (BOD) "wired" with PAA-PVI-[Os(4,4'-dichloro-2,2'-bipyridine)2Cl]+/2+ (polymer II), having a redox potential of + 0.36 V versus Ag/AgCl. The ring-disk electrode was held facing up, and a 30-microL drop was placed on it for the assay, with the ring poised at -0.3 V/ AgAgCl and the disk poised at -0.1 V/ Ag/AgCl. Even though the atmosphere over the drop was O2 at 1 atm pressure, the wired BOD disk scavenged the O2 so effectively that the glucose-reduced FADH2 of GOx was not oxidized by O2, the natural cosubstrate of the enzyme.     

Redox mediation and photomechanical oscillations involving photosensitive cyclometalated Ru(II) complexes, glucose oxidase, and peroxidase

Intact photosensitive cyclometalated RuII derivatives of 2-phenylpyridine or N,N-dimethylbenzylamine cis-[Ru-(C approximately N)(LL)X2]PF6 [C approximately N = o-C6H4-py or o-C6H4CH2NMe2; LL = 1,10-phenanththroline (phen), 2,2'-bipyridine (bpy), or 4,4'-Me2-2,2'-bipyridine (Me2bpy); X = MeCN or pyridine (py)] are efficient mediators of glucose oxidase (GO) from Aspergillus niger and horseradish peroxidase (HRP). Their redox potentials in an aqueous buffer are in the range 0.15-0.35 V versus SCE, and the rate constants for the oxidation GO(red) (where red indicates reduced) by the electrochemically generated RuIII species equal (1.7-2.5) x 10(6) M(-1) s(-1) at pH 7 and 25 degrees C. The redox potentials of all complexes decrease cathodically by 0.4-0.6 V upon irradiation by visible light because of the photoinduced solvolysis of acetonitrile or py ligands. These in situ generated species display an even better mediating performance with HRP, although their behavior toward GO is different. The loading of a ruthenium unit into the protein interior brings about large catalytic currents in a self-assembled system GO-Ru-D-glucose. The estimated rate constant for intramolecular electron transfer from FADH2 of the active site at RuIII, k(intra), equals 4.4 x 10(3) s(-1). This suggests that the distance between the redox partners is around 19 A. The value of 21 A was obtained through the docking analysis of a possible closest-to-FAD localization of a Ru-containing fragment derived from the irradiated complex cis-[Ru(o-C6H4-py)-(phen)(MeCN)2]PF6. The operational stability of the GO-Ru assemblies depends on the nature of complex used, the highest being observed for cis-[Ru(o-C6H4-py)(Me2-bpy)(MeCN)2]PF6 (2). UV-vis studies of interaction of 2 with GO revealed photomechanical oscillations in the system GO-Ru-D-glucose. When irradiated complex 2 is mixed with GO and D-glucose, the absorbance at 510 nm increases because of the enzymatic reduction of RuIII to RuII. The absorbance drops rapidly and then increases as in the first cycle after shaking the reaction solution. Many cycles are possible, and the rate of absorbance increase does not depend on a cycle number. A plausible mechanism of the oscillations is presented.     

Electrochemical DNA sensors based on enzyme dendritic architectures: an approach for enhanced sensitivity

The modification of enzymes with multiple single-stranded oligonucleotides opens up a new concept for the development of DNA sensors with enhanced sensitivity. This work describes the generation of reporter sequences labeled with an enzyme for the demonstration of their ability to specifically hybridize and to permit signal amplification by successive hybridization steps. The synthetic pathway for the labeling of GOx with oligonucleotide sequences is based on the oxidation of the glycosidic residues of the enzyme and their covalent binding with 5'-end amine-modified oligonucleotides. Spectrophotometric characterization of these functionalized sequences results in an average number of three linked oligonucleotides per enzyme molecule. Their specificity is demonstrated in both a direct and a sandwich-type hybridization assay. The transduction of the enzyme-linked DNA sensors is based on self-assembled multilayers, including a chemically modified anionic horseradish peroxidase electrochemically connected to a water-soluble cationic poly[(vinylpyridine)Os(bpy)(2)Cl] redox polymer in an electrostatic ordered assembly. The sensing layer is constructed by the covalent binding of the DNA probe over the redox polymer through the 3'-phosphate group, enabling the capture of the target sequence. Upon addition of glucose, hybridization results in the production of H(2)O(2), which readily diffuses to the electrocatalytic assembly, giving rise to a cathodic current at 100 mV vs Ag/AgCl. Hybridization is always performed at room temperature, and after 30 min of incubation, an amperometric response is obtained that is proportional to DNA concentration. The simultaneous sandwich assay enables the quantification of a free-label 44-mer oligonucleotide at 1 nM concentration. Signal amplification is realized by a new hybridization step over the free sequences, giving rise to a dendritic architecture that accumulates enzyme molecules per hybridization event.     

Redox polymer and probe DNA tethered to gold electrodes for enzyme-amplified amperometric detection of DNA hybridization

The detection of nucleic acids based upon recognition surfaces formed by co-immobilization of a redox polymer mediator and DNA probe sequences on gold electrodes is described. The recognition surface consists of a redox polymer, [Os(2,2'-bipyridine)2(polyvinylimidazole)(10)Cl](+/2+), and a model single DNA strand cross-linked and tethered to a gold electrode via an anchoring self-assembled monolayer (SAM) of cysteamine. Hybridization between the immobilized probe DNA of the recognition surface and a biotin-conjugated target DNA sequence (designed from the ssrA gene of Listeria monocytogenes), followed by addition of an enzyme (glucose oxidase)-avidin conjugate, results in electrical contact between the enzyme and the mediating redox polymer. In the presence of glucose, the current generated due to the catalytic oxidation of glucose to gluconolactone is measured, and a response is obtained that is binding-dependent. The tethering of the probe DNA and redox polymer to the SAM improves the stability of the surface to assay conditions of rigorous washing and high salt concentration (1 M). These conditions eliminate nonspecific interaction of both the target DNA and the enzyme-avidin conjugate with the recognition surfaces. The sensor response increases linearly with increasing concentration of target DNA in the range of 1 x 10(-9) to 2 x 10(-6) M. The detection limit is approximately 1.4 fmol, (corresponding to 0.2 nM of target DNA). Regeneration of the recognition surface is possible by treatment with 0.25 M NaOH solution. After rehybridization of the regenerated surface with the target DNA sequence, >95% of the current is recovered, indicating that the redox polymer and probe DNA are strongly bound to the surface. These results demonstrate the utility of the proposed approach.     

Electrostatic electrochemistry at insulators

The identity of charges generated by contact electrification on dielectrics has remained unknown for centuries and the precise determination of the charge density is also a long-standing challenge. Here, electrostatic charges on Teflon (polytetrafluoroethylene) produced by rubbing with Lucite (polymethylmethacrylate) were directly identified as electrons rather than ions by electrochemical (redox) experiments with charged Teflon used as a single electrode in solution causing various chemical reactions: pH increases; hydrogen formation; metal deposition; Fe(CN)(6)(3-) reduction; and chemiluminescence in the system of Teflon(-)/Ru(bpy)(3)(2+)/S(2)O(8)(2-) (analogous to electrogenerated chemiluminescence). Moreover, copper deposition could be amplified by depositing Pd first in a predetermined pattern, followed by electroless deposition to produce Cu lines. This process could be potentially important for microelectronic and other applications because Teflon has desirable properties including a low dielectric constant and good thermal stability. Charge density was determined using Faraday's law and the significance of electron transfer processes on charged polymers and potentially other insulators have been demonstrated.     

Deposition of α-SiΜο12Ο40-[Ru(bipyridine)(terpyridine)Cl] multilayer film on single wall carbon nanotube modified glassy carbon electrode: Improvement of the electrochemical properties and chemical stability

A simple procedure was developed for the preparation of glassy carbon electrodes modified with single wall carbon nanotubes (SWCNTs) and multilayers of SiΜο₁₂Ο₄₀⁴⁻-[Ru(bpy)(tpy)Cl]⁺(byp; bipyridine, tpy; terpyridine). Layer-by-layer deposition technique was used for the multilayer formation of SiΜο₁₂Ο₄₀⁴⁻-[Ru(bpy)(tpy)Cl]⁺ onto SWCNTs films. Based on the strong electrostatic attraction of oppositely charged species a Ru-complex/poly oxometalate hybrid film strongly and irreversibly adsorbed on the glassy carbon electrode modified with single walled carbon nanotubes. The multilayer assembly exhibited good stability and excellent electrochemical reversibility for both redox systems in the pH range1-7. It was found that up to fifteen monolayers could be deposited onto a carbon nanotube film with well defined redox behavior. The modified electrode shows excellent electrocatalytic activity towards sulfite oxidation. Due to synergistic effect between SWCNTs and oppositely charged species the repeated alternate adsorption of anions and cations by this simple dipping method leads to molecular sandwiches with interesting redox activity and remarkable stability.     

Amperometric glucose microelectrodes prepared through immobilization of glucose oxidase in redox hydrogels.

Glucose microelectrodes have been formed with glucose oxidase immobilized in poly[(vinylpyridine)Os(bipyridine)2Cl] derivative-based redox hydrogels on beveled carbon-fiber microdisk (7 microns diameter) electrodes. In the resulting microelectrode, the steady-state glucose electrooxidation current density is 0.3 mA cm-2 and the sensitivity is 20 mA cm-2 M-1. The current density and sensitivity are 10 times higher than in macroelectrodes made with the same hydrogel. Furthermore, the current is less affected by a change in the partial pressure of oxygen. The higher current density and lower oxygen sensitivity point to the efficient collection of electrons through their diffusion in the redox hydrogel to the electrode surface. These results contrast with those observed for enzyme electrodes based on diffusing mediators, where loss of the enzyme-reduced mediator by radial diffusion to the solution decreases the current densities of microelectrodes relative to similar macroelectrodes.     

Electrochemiluminescence from Os(phen)2(dppene)2+ (phen = 1,10-phenanthroline and dppene = bis(diphenylphosphino)ethene)

The electrochemiluminescence (ECL) of Os(phen)2(dppene)2+ (phen = 1,10-phenanthroline and dppene = bis(diphenylphosphino)ethene) is reported in mixed CH3CN/H2O (50:50 v/v) and aqueous (0.1 M KH2PO4) solutions with tri-n-propylamine (TPrA) as an oxidative-reductive coreactant. ECL efficiencies (phi(ecl) = photons emitted/redox event) of 2.0 in aqueous, and 0.95 in mixed for Os(phen)2(dppene)2+ were obtained using Ru(bpy)3(2+) (bpy = 2,2'-bipyridine) as a relative standard (phi(ecl) = 1). Photoluminescence (PL) efficiencies of 0.094 and 0.053 were obtained in aqueous and mixed solutions, respectively, as compared to Ru(bpy)3(2+) (phi(em) = 0.042). The ECL spectra were identical to photoluminescence spectra (lambda(max) approximately 584 nm), indicating formation of the same metal-to-ligand (MLCT) excited states in both ECL and PL. The ECL is linear over several orders of magnitude in aqueous and mixed solution, with theoretical detection limits (blank plus three times the standard deviation of the noise) of 16.9 nM in H2O and 0.29 nM in CH3CN/H2O (50:50 v/v).     

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.