Simultaneous direct electrochemiluminescence and catalytic voltammetry detection of DNA in ultrathin films

Direct electrochemiluminescence (ECL) involving DNA was demonstrated in 10 nm films of cationic polymer [Ru(bpy)(2)(PVP)(10)](2+) assembled layer-by-layer with DNA. A square wave voltammetric waveform oxidized the Ru(II) sites in the metallopolymer to Ru(III), and ECL was measured simultaneously with catalytic voltammetric peaks in a simple apparatus. Significant ECL generation occurred only when guanine bases were present on oligonucleotides in the films. This result along with knowledge of proposed ECL pathways suggests that guanine radicals initially formed by catalytic oxidation of guanines by Ru(III) react with the metallopolymer to produce electronically exited Ru(II) sites in the film. ECL and catalytic SWV peaks were sensitive to oligonucleotide hybridization and chemical DNA damage. Simultaneous linear growth of ECL and SWV peaks occurred after incubation with known DNA damage agent styrene oxide over 20 min. The estimated detection limit was 1 damaged DNA base in 1000. Control incubations of metallopolymer/ds-DNA films in buffer containing unreactive toluene resulted in no significant changes of the ECL or SWV peaks.     

Photoinduced energy- and electron-transfer processes in dinuclear Ru(II)-Os(II), Ru(II)-Os(III), and Ru(III)-Os(II) trisbipyridine complexes containing a shape-persistent macrocyclic spacer.

The PF6- salt of the dinuclear [(bpy)2Ru(1)Os(bpy)2]4+ complex, where 1 is a phenylacetylene macrocycle which incorporates two 2,2'-bipyridine (bpy) chelating units in opposite sites of its shape-persistent structure, was prepared. In acetonitrile solution, the Ru- and Os-based units display their characteristic absorption spectra and electrochemical properties as in the parent homodinuclear compounds. The luminescence spectrum, however, shows that the emission band of the Ru(II) unit is almost completely quenched with concomitant sensitization of the emission of the Os(II) unit. Electronic energy transfer from the Ru(II) to the Os(II) unit takes place by two distinct processes (k(en) = 2.0x10(8) and 2.2x10(7) s(-1) at 298 K). Oxidation of the Os(II) unit of [(bpy)2Ru(1)Os(bpy)2]4+ by Ce(IV) or nitric acid leads quantitatively to the [(bpy)2Ru(II)(1)Os(III)(bpy)2]5+ complex which exhibits a bpy-to-Os(III) charge-transfer band at 720 nm (epsilon(max) = 250 M(-1) cm(-1)). Light excitation of the Ru(II) unit of [(bpy)2Ru(II)(1)Os(III)(bpy)2]5+ is followed by electron transfer from the Ru(II) to the Os(III) unit (k(el,f) = 1.6x10(8) and 2.7x10(7) s(-1)), resulting in the transient formation of the [(bpy)2Ru(III)(1)Os(II)(bpy)2]5+ complex. The latter species relaxes to the [(bpy)2Ru(II)(1)Os(III)(bpy)2]5+ one by back electron transfer (k(el,b) = 9.1x10(7) and 1.2x10(7) s(-1)). The biexponential decays of the [(bpy)2*Ru(II)(1)Os(II)(bpy)2]4+, [(bpy)2*Ru(II)(1)Os(III)(bpy)2]5+, and [(bpy)2Ru(III)(1)Os(II)(bpy)2]5+ species are related to the presence of two conformers, as expected because of the steric hindrance between hydrogen atoms of the pyridine and phenyl rings. Comparison of the results obtained with those previously reported for other Ru-Os polypyridine complexes shows that the macrocyclic ligand 1 is a relatively poor conducting bridge.     

Electrochemiluminescence of Ru(II) complexes immobilized on a magnetic microbead surface: distribution of magnetic microbeads on the electrode surface and effect of azide ion

The electrochemiluminescence (ECL) of magnetic microbeads modified with tris(2,2'-bipyridine)ruthenium(II) ([Ru(bpy)3]2+) was studied in the presence of tri-n-propylamine (TPA) to develop highly sensitive ECL detection system, where the employed microbead has a diameter of 4.5 microm. The ECL signal of the [Ru(bpy)3]2+ derivative-modified magnetic microbeads was found to be affected by the geometrical distribution of the magnetic microbeads on the electrode surface. The ECL peak intensity increased with increasing the number of the beads on the electrode surfaces up to 1.6 x 10(6) beads cm(-2), although above 1.6 x 10(6) beads cm(-2), it decreased. The ECL decrease arises from the physical prevention of the ECL from reaching the photomultiplier tube by the excessive beads. The observed peak ECL signal of the [Ru(bpy)3]2+ derivative-modified magnetic microbeads in the presence of NaN3, which serves as a preservative substance, mainly appeared at a potential of +0.90 V vs Ag/AgCl where [Ru(bpy)3]2+ is hardly oxidized, whereas the ECL signal in the absence of NaN3 appeared at a potential of +1.15 V. The presence of NaN3 on the electrode surface retards formation of an oxide layer on the electrode surfaces and promotes TPA oxidation. The ECL response at +0.90 V was mainly attributed to ECL reaction of excited-state [Ru(bpy)3]2+* formed by oxidation of [Ru(bpy)3]+ with TPA radical cation, where the [Ru(bpy)3]+ was generated by reduction of [Ru(bpy)3]2+ with TPA radical.     

Binding sites of [Ru(bpy)2(H2O)2](BF4)2 with sulfur- and histidine-containing peptides studied by electrospray ionization mass spectrometry and tandem mass spectrometry

The composition and binding sites of cis-[Ru(II)(bpy)2]2+-bound sulfur-containing peptides of Met-Arg-Phe-Ala, glutathione and oxidized glutathione, and also histidine-containing peptide of oxidized insulin B chain, were investigated by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS). The composition of Ru(II)-containing peptides was precisely determined by ESI-MS, zoom scan and simulation of isotope distribution patterns. MS/MS analysis shows that, in sulfur-containing peptides, the Ru(II) complex prefers to anchor to a carboxyl group, although some other potential binding sites of thiol, thioether and N-terminal amino groups present in these peptides, and in oxidized insulin B chain, Ru(II) first anchors to His10, then either to the hydroxyl group of Thr27 or to the carboxyl group of Ala30. Its secondary structure and microenvironment surrounding the potential binding sites may affect the binding ability of cis-[Ru(II)(bpy)2]2+ to oxidized insulin B chain.     

Electrolyte Effects on Charge Transport Behavior of [Os(bpy)2(PVP)10Cl]Cl and [Ru(bpy)2(PVP)10Cl]Cl Redox Polymers in Ultra‐Thin Films of Polyions

Metallopolymer films have important applications in electrochemical catalysis. The alternate electrostatic layer‐by‐layer method was used to assemble films of [Ru(bpy)₂(PVP)₁₀Cl]Cl (denoted as ClRu‐PVP) and [Os(bpy)₂(PVP)₁₀Cl]Cl (ClOs‐PVP) metallopolymers onto pyrolytic graphite electrodes. Film thickness estimated by quartz crystal microbalance was 6–8 nm. The effects of pH, electrolyte species and concentration on the electrochemical properties of these electroactive polymers were studied using cyclic voltammetry (CV). Behavior in various electrolytes was compared. Also the mass changes within the ultra‐thin film during redox of Os^2+/3+ were characterized by in situ electrochemical quartz crystal microbalance (EQCM). The results indicate rapid reversible electron transfer, and show that both ClRu‐PVP and ClOs‐PVP have compact surface structures while ClOs‐PVP is a little denser than ClRu‐PVP. Although hydrogen ions do not participate in the chemical reaction of either film, the movement of Na⁺ cation and water accompanies the redox process of ClOs‐PVP films.     

Surface confinement and its effects on the luminescence quenching of a ruthenium-containing metallopolymer

Luminescence quenching of the metallopolymers [Ru(bpy)(2)(PVP)(10)](2+) and [Ru(bpy)(2)(PVP)(10)Os(bpy)(2)](4+), both in solution and as thin films, is reported, where bpy is 2,2'-bipyridyl and PVP is poly(4-vinylpyridine). When the metallopolymer is dissolved in ethanol, quenching of the ruthenium excited state, Ru(2+*), within [Ru(bpy)(2)(PVP)(10)](2+) by [Os(bpy)(3)](2+) proceeds by a dynamic quenching mechanism and the rate constant is (1.1 +/- 0.1) x 10(11) M(-1) s(-1). This quenching rate is nearly two orders of magnitude larger than that found for quenching of monomeric [Ru(bpy)(3)](2+) under the same conditions. This observation is interpreted in terms of an energy transfer quenching mechanism in which the high local concentration of ruthenium luminophores leads to a single [Os(bpy)(3)](2+) centre quenching the emission of several ruthenium luminophores. Amplifications of this kind will lead to the development of more sensitive sensors based on emission quenching. Quenching by both [Os(bpy)(3)](2+) and molecular oxygen is significantly reduced within a thin film of the metallopolymer. Significantly, in both optically driven emission and electrogenerated chemiluminescence, emission is observed from both ruthenium and osmium centres within [Ru(bpy)(2)(PVP)(10)Os(bpy)(2)](4+) films, i.e. the ruthenium emission is not quenched by the coordinated [Os(bpy)(2)](2+) units. This observation opens up new possibilities in multi-analyte sensing since each luminophore can be used to detect separate analytes, e.g. guanine and oxoguanine.     

钌(II)多吡啶配合物的合成、荧光性质及与脱氧核糖核酸DNA的 作用机制研究

设计合成含多个配位中心的多吡啶配体ODCIP (3,4-二氯基苯并咪唑并[4,5-f][1,10]邻菲咯啉)及其钌(II)多吡啶配合物[Ru(bpy)2ODCIP]2＋. 运用元素分析、红外光谱、核磁谱和质谱对配体及配合物进行结构表征. 利用紫外吸收光谱、荧光光谱和粘度法研究了[Ru(bpy)2ODCIP]2＋与DNA(脱氧核糖核酸)的作用机制、与Co2＋配位后与DNA的作用机制及其荧光变化情况. 结果表明[Ru(bpy)2ODCIP]2＋与DNA通过部分插入模式作用, [Ru(bpy)2ODCIP]2＋与Co2＋配位形成的双核配合物[Ru(bpy)2(ODCIP)Co]4＋也能与DNA插入结合. 进一步利用稳态荧光发射光谱、荧光淬灭实验等方法研究了单核配合物[Ru(bpy)2ODCIP]2＋和双核配合物[Ru(bpy)2(ODCIP)Co]4＋的荧光性质.     

Mechanistic implications of proton transfer coupled to electron transfer.

The kinetics of electron transfer for the reactions cis-[Ru(IV)(bpy)2(py)(O)]2+ + H+ + [Os(II)(bpy)3]2+ <==> cis-[Ru(III)(bpy)2(py)(OH)]2+ + [Os(III)(bpy)3]3+ and cis-[Ru(III)(bpy)2(py)(OH)]2+ + H+ + [Os(II)(bpy)3]2+ <==> cis-[Ru(II)(bpy)2(py)(H2O)]2+ + [Os(III)(bpy)3]3+ have been studied in both directions by varying the pH from 1 to 8. The kinetics are complex but can be fit to a double "square scheme" involving stepwise electron and proton transfer by including the disproportionation equilibrium, 2cis-[Ru(III)(bpy)2(py)(OH)]2+ <==> (3 x 10(3) M(-1) x s(-1) forward, 2.1 x 10(5) M(-1) x s(-1) reverse) cis-[Ru(IV)(bpy)2(py)(O)]2+ + cis-[Ru(II)(bpy)2(py)(H2O)]2+. Electron transfer is outer-sphere and uncoupled from proton transfer. The kinetic study has revealed (1) pH-dependent reactions where the pH dependence arises from the distribution between acid and base forms and not from variations in the driving force; (2) competing pathways involving initial electron transfer or initial proton transfer whose relative importance depends on pH; (3) a significant inhibition to outer-sphere electron transfer for the Ru(IV)=O2+/Ru(III)-OH2+ couple because of the large difference in pK(a) values between Ru(IV)=OH3+ (pK(a) < 0) and Ru(III)-OH2+ (pK(a) > 14); and (4) regions where proton loss from cis-[Ru(II)(bpy)2(py)(H2O)]2+ or cis-[Ru(III)(bpy)2(py)(OH)]2+ is rate limiting. The difference in pK(a) values favors more complex pathways such as proton-coupled electron transfer.     

Comparative study of ruthenium(II) tris(bipyridine) derivatives for electrochemiluminescence application

Several [Ru(bpy)3]2+ (bpy = 2,2'-bipyridine and its derivatives) complexes were synthesized and compared electrochemically and spectroscopically in the search for better luminophores for electrochemiluminescence (ECL)-based analytical applications. ECL measurement in [Ru(bpy)3]2+/tripropylamine (TPA) aqueous buffer solutions has led to a conclusion that due to the complexity of the ECL generation process, the photoluminescence efficiency cannot be used to predict ECL intensity and there is no obvious relationship between the photoluminescence quantum yield and the ECL intensity. Under the present experimental condition, when compared with the pristine [Ru(bpy)3]2+, the ethoxycarbonyl-substituted derivative, [Ru(bpy-COOEt)3]2+, one of the most efficient luminophores under photoexcitation, did not generate reasonably intense ECL, whereas luminophores with lower photoluminescence quantum yields demonstrated higher ECL. These findings are useful for further efforts in the search for more efficient ECL luminophores.     

Synthesis and Fluorescence of a New Active Material for Electrochemiluminescent Sensor:Ru(phen)2[phen-NHCO(CH2)4Br](PF6)2

Recently, much attention has been paid to Ru(II) complexes because of their excellent properties of photochemistry, phtophysis. Bis(2,2'-bipyridine)[4-methyl-4'-(6-bromohexyl)-2,2'-bipyridine] ruthenium(II) perchlorate has been used as an active material for electrochemiluminescent (ECL) sensor for selective detection of oxalic acid.It is known that ECL efficiency of Ru(phen)₃²⁺ is much higher than that of Ru(bpy)₃²⁺. In order to make out more efficient ECL sensor, we have designed and synthesized a new Ru(II) complex, Ru(phen)₂[phen-NHCO(CH₂)₄Br](PF₆)₂.     

DNA mismatch detection by resonance energy transfer between ruthenium(II) and osmium(II) tris(2,2'-bipyridyl) chromophores

Octahedral tris-chelate complexes [M(II)(bpy)(3)](2+) (M = Ru or Os, bpy = 2,2'-bipyridyl), covalently attached to the 3'- and 5'-phosphates of two oligonucleotides, are juxtaposed when hybridized contiguously to a fully complementary DNA target. Visible metal-to-ligand charge-transfer (MLCT) excitation of the [Ru(II)(bpy)(3)](2+) unit leads to resonance energy transfer to the MLCT state of the [Os(II)(bpy)(3)](2+) moiety, with the energy transfer efficiency depending on the degree of hybridization. The extent of attenuation of the intense red luminescence from the Ru(II) chromophore hence allows highly sensitive structural probing of the assembly and constitutes a novel approach to DNA sensing which is capable of detecting mutations.     

Photobiological impact of [{(bpy)2Ru(dpp)}2RhCl2]Cl5 and [{(bpy)2Os(dpp)}2RhCl2]Cl5 [bpy=2,2'-bipyridine; dpp=2,3-Bis(2-pyridyl)pyrazine] on vero cells

The in vitro photobiology of the supramolecular complexes [{(bpy)2Ru(dpp)}2RhCl2]Cl5 and [{(bpy)2Os(dpp)}2RhCl2]Cl5 [bpy=2,2'-bipyridine; dpp=2,3-bis(2-pyridyl)pyrazine] with African green monkey kidney epithelial (Vero) cells was investigated. Previously, the complexes have been shown to photocleave DNA in the presence or absence of O2. Vero cell replication was uninhibited for cells exposed to the metal complex but protected from light. Vero cells that were exposed to metal complex, rinsed, and illuminated with >460 nm light showed a replication response that was metal complex concentration-dependent. Vero cells exposed to 3.0-120 microM [{(bpy)2Ru(dpp)}2RhCl2]Cl5 and illuminated showed inhibition of cell growth, with evidence of cell death seen for complex concentrations>or=10 microM. Cells exposed to [{(bpy)2Os(dpp)}2RhCl2]Cl5 at concentrations of 5.5-110 microM, rinsed, and illuminated showed only inhibition of cell growth. The impact of [{(bpy)2Ru(dpp)}2RhCl2]Cl5 and [{(bpy)2Os(dpp)}2RhCl2]Cl5 on cell growth following illumination shows the promise of this new structural motif as a photodynamic therapy agent.     

Nafion-stabilized magnetic nanoparticles (Fe3O4) for [Ru(bpy)3]2+(bpy = bipyridine) electrogenerated chemiluminescence sensor

A highly sensitive and stable [Ru(bpy)3]2+ ECL sensor has been fabricated based on the multilayer films of Nafion-stabilized magnetic nanoparticles (Nafion/Fe3O4) formed on a platinum electrode surface by means of an external magnet.     

Multisite effects on intervalence charge transfer in a clusterlike trinuclear assembly containing ruthenium and osmium

The intervalence charge transfer (IVCT) properties of the mixed-valence forms of the diastereoisomers of the dinuclear [[Ru(bpy)2](mu-HAT)[M(bpy)2]]5+ (M = Ru or Os) complexes and the trinuclear heterochiral [[Ru(bpy)2]2[Os(bpy)2](mu-HAT)]n+ (n = 7, 8; HAT = 1,4,5,8,9,12-hexaazatriphenylene; bpy = 2,2'-bipyridine) species display a marked dependence on the nuclearity and extent of oxidation of the assemblies, while small differences are also observed for the diastereoisomers of the same complex in the dinuclear cases. The mixed-valence heterochiral [[Ru(bpy)2]2[Os(bpy)2](mu-HAT)]n+ (n = 7, 8) forms exhibit IVCT properties that are intermediate between those of the diastereoisomeric forms of the localized hetero-dinuclear complex [[Ru(bpy)2](mu-HAT)[Os(bpy)2]]5+ and the borderline localized-to-delocalized homo-trinuclear complex [[Ru(bpy)2]3(mu-HAT)]n+ (n = 7, 8). The near-infrared (NIR) spectrum of the +7 mixed-valence species exhibits both interconfigurational (IC) and IVCT transitions which are quantitatively similar to those in [[Ru(bpy)2](mu-HAT)[Os(bpy)2]]5+ and are indicative of the localized mixed-valence formulation [[Ru(II)(bpy)2]2[Os(III)(bpy)2](mu-HAT)]7+. The +8 state exhibits a new band attributable to an IVCT transition in the near-infrared region.     

[Ru(bpy)_2tpphz]~(2+)与Zn~(2+)形成的双核配合物的荧光性质研究

近年来,钌多吡啶配合物与DNA的作用得到了比较广泛的研究,并且发展了一系列具有特定功能的钌配合物犤1犦。如传统的DNA分子光开关犤Ru(bpy)2dppz犦2+和犤Ru(phen)2dppz犦2+犤2,3犦(bpy=2,2'-联吡啶,phen=1,10-菲咯啉,dppz=二吡啶犤3,2-a:2',3'-c犦吩嗪)。这些配合物与DNA具有较强的结合力,在水溶液中几乎不发光,但在DNA存在下则有强烈荧光发出。这是由于配合物插入DNA的碱基对之后,保护了dppz的吡嗪环上的N原子,使其免受水分子的进攻从而导致配合物荧光的恢复。但是对于大多数的多吡啶钌配合物来讲,由于其自身较强的背景荧光或与DN…     

流动体系中维生素B~1的电致化学发光研究

在0.1mol/LNaOH水溶液中(pH12.6),维生素B~1的水解产物在玻碳电极上于+0.88V(vs.Ag/AgCl,下同)处被氧化,其氧化产物在+1.20V处与被氧化的Ru(bpy)~3^2^+反应,生成激发态的Ru(bpy)~3^2^+^*而发光,发光波长为609nm,研究结果表明水溶液的pH值影响了维生素B~1的水解速率,从而引起发光强度的明显差异。     

Nanoscale-enhanced Ru(bpy)3(2+) electrochemiluminescence labels and related aptamer-based biosensing system

A unique multilabeling at a single-site protocol of the Ru(bpy)(3)(2+) electrochemiluminescence (ECL) system is proposed. Nanoparticles (NPs) were used as assembly substrates to enrich ECL co-reactants of Ru(bpy)(3)(2+) to construct nanoscale-enhanced ECL labels. Two different kinds of NP substrates [including semiconductor NPs (CdTe) and noble metal NPs (gold)] capped with 2-(dimethylamino)ethanethiol (DMAET) [a tertiary amine derivative which is believed to be one of the most efficient of co-reactants of the Ru(bpy)(3)(2+) system] were synthesized through a simple one-pot synthesis method in aqueous media. Although both CdTe and gold NPs realized the enrichment of ECL co-reactants, they presented entirely different ECL performances as nanoscale ECL co-reactants of Ru(bpy)(3)(2+). The different effects of these two NPs on the ECL of Ru(bpy)(3)(2+) were studied. DMAET-capped CdTe NPs showed enormous signal amplification of Ru(bpy)(3)(2+) ECL, whereas DMAET-capped gold NPs showed a slight quenching effect of the ECL signal. DMAET-capped CdTe NPs can be considered to be excellent nanoscale ECL labels of the Ru(bpy)(3)(2+) system, as even a NP solution sample of 10(-18) M was still detectable after an electrostatic self-assembly concentration process. DMAET-capped CdTe NPs were further applied in the construction of aptamer-based biosensing system for proteins and encouraging results were obtained.     

Tris(2,2′-bipyridyl)ruthenium(II) electrogenerated chemiluminescence sensor based on carbon nantube dispersed in sol-gel-derived titania-Nafion composite films

A highly sensitive and stable tris(2,2′-bipyridyl)ruthenium(II) (Ru(bpy)₃²⁺) electrogenerated chemiluminescence (ECL) sensor was developed based on carbon nanotube (CNT) dispersed in mesoporous composite films of sol-gel titania and perfluorosulfonated ionomer (Nafion). Single-wall (SWCNT) and multi-wall carbon nanotubes (MWCNT) can be easily dispersed in the titania-Nafion composite solution. The hydrophobic CNT in the titania-Nafion composite films coated on a glassy carbon electrode certainly increased the amount of Ru(bpy)₃²⁺ immobilized in the ECL sensor by adsorption of Ru(bpy)₃²⁺ onto CNT surface, the electrocatalytic activity towards the oxidation of hydrophobic analytes, and the electronic conductivity of the composite films. Therefore, the present ECL sensor based on the CNT-titania-Nafion showed improved ECL sensitivity for tripropylamine (TPA) compared to the ECL sensors based on both titania-Nafion composite films without CNT and pure Nafion films. The present Ru(bpy)₃²⁺ ECL sensor based on the MWCNT-titania--Nafion composite gave a linear response (R² = 0.999) for TPA concentration from 50 nM to 1.0 mM with a remarkable detection limit (S/N = 3) of 10 nM while the ECL sensors based on titania-Nafion composite without MWCNT, pure Nafion films, and MWCNT-Nafion composite gave a detection limit of 0.1 μM, 1 μM, and 50 nM, respectively. The present ECL sensor showed outstanding long-term stability (no signal loss for 4 months).     

Ru(bpy)3]2+-doped silica nanoparticles within layer-by-layer biomolecular coatings and their application as a biocompatible electrochemiluminescent tag material

[Ru(bpy)3]2+-doped silica (RuSi) nanoparticles were synthesized by using a water/oil microemulsion method. Stable electrochemiluminescence (ECL) was obtained when the RuSi nanoparticles were immobilized on a glassy carbon electrode by using tripropylamine (TPA) as a coreactant. Furthermore, the ECL of the RuSi nanoparticles with layer-by-layer biomolecular coatings was investigated. Squential self-assembly of the polyelectrolytes and biomolecules on the RuSi nanoparticles gave nanocomposite suspensions, the ECL of which decreased on increasing the number of bilayers. Moreover, factors that affected the assembly and ECL signals were investigated. The decrease in ECL could be assigned to steric hindrance and limited diffusion of the coreactant molecules in the silica matrix after they were attached to the biomolecules. Since surface modification of the RuSi nanoparticles can improve their biocompatibility and prevent leaking of the [Ru(bpy)3]2+ ions, the RuSi nanoparticles can be readily used as efficient and stable ECL tag materials in immunoassay and DNA detection.     

DNA biosensor based on the electrochemiluminescence of Ru(bpy)3(2+) with DNA-binding intercalators

This paper reports a novel detection method for DNA hybridization based on the electrochemiluminescence (ECL) of Ru(bpy)(3)(2+) with a DNA-binding intercalator as a reductant of Ru(bpy)(3)(3+). Some ECL-inducible intercalators have been screened in this study using electrochemical methods combined with a chemiluminescent technique. The double-stranded DNA intercalated by doxorubicin, daunorubicin, or 4',6-diamidino-2-phenylindole (DAPI) shows a good ECL with Ru(bpy)(3)(2+) at +1.19 V (versus Ag/AgCl), while the non-intercalated single-stranded DNA does not. In order to stabilize the self-assembled DNA molecules during ECL reaction, we constructed the ECL DNA biosensor separating the ECL working electrode with an immobilized DNA probe. A gold electrode array on a plastic plate was assembled with a thru-hole array where oligonucleotide probes were immobilized in the side wall of thru-hole array. The fabricated ECL DNA biosensor was used to detect several pathogens using ECL technique. A good specificity of single point mutations for hepatitis disease was obtained by using the DAPI-intercalated Ru(bpy)(3)(2+) ECL.