Electrochemical behavior and detection of hepatitis B virus DNA PCR production at gold electrode

Sequence-known short-stranded hepatitis B virus (HBV) DNA fragment (181 bps) was obtained by PCR method. The strategy for its electrochemical detection was designed by covalently immobilizing single-stranded HBV DNA on gold electrode surface via carboxylate ester as a linkage between 3′-hydroxy end of DNA and carboxyl group of thioglycolic acid (TGA) self-assembled monolayer. The hybridization reaction on surface was evidenced by electrochemical methods using ferrocenium hexafluorophosphate (FcPF₆) as an electroactive indicator. The interactions of Fc⁺ with single-stranded (ss) and double-stranded (ds) HBV DNA immobilized on TGA monolayer were studied. The difference between the responses of Fc⁺ at ss- and ds-DNA/Au electrodes suggested that this hybridization biosensor could be conveniently used to monitor DNA hybridization with a high sensitivity. AC impedance and XPS techniques have been employed to characterize the immobilization of ss-DNA on the gold surface.     

Electrochemical DNA sensing using osmium complexes as hybridization indicators

A surface-based method for the study of the interactions of DNA with redox-active osmium complexes is described. The study was carried out using gold electrodes modified with DNA by adsorption and [Os(bpy)3]3+/2+ (bpy=2,2'-bipyridyl) or [Os(phen)3]3+/2+ (phen=1,10-phenantroline) as electrochemical indicators. The method, which is simple and reagent saving, allows the accumulation of osmium complexes on the DNA layer. The amount of osmium complex bound by the layer of double-stranded (dsDNA) or single-stranded DNA (ssDNA) adsorbed at gold electrodes was estimated from the cyclic voltammetric (CV) peak charge of osmium complex reduction. The dissociation constants (K) for the oxidized and reduced forms of a bound species are also estimated. [Os(phen)3]3+/2+ was applied to a probe for electrochemical DNA sensing. A thiol-linked single-stranded DNA probe was immobilized through the S-Au bonding to 70 pmol/cm2 on a gold electrode. Following hybridization with the complementary DNA, the osmium complex was electrochemically accumulated on the double-stranded DNA layer and the differential pulse voltammogram for this electrode gave an electrochemical signal due to the redox reaction of [Os(phen)3]3+/2+ that was bound to the double-stranded DNA on the electrode.     

A nucleic acid biosensor for the detection of a short sequence related to the hepatitis B virus using bis(benzimidazole)cadmium(II) dinitrate as an electrochemical indicator

A novel hybridization indicator, bis(benzimidazole)cadmium(II) dinitrate (Cd(bzim)(2)(NO(3))(2)), was utilized to develop an electrochemical DNA biosensor for the detection of a short DNA sequence related to the hepatitis B virus (HBV). The sensor relies on the immobilization and hybridization of the 21-mer single-stranded oligonucleotide from the HBV long repeat at the glassy carbon electrode (GCE). The hybridization between the probe and its complementary sequence as the target was studied by enhancement of the peak of the Cd(bzim)(2)(2+) indicator using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). Numerous factors affecting the probe immobilization, target hybridization, and indicator binding reactions were optimized to maximize the sensitivity and speed of the assay time. With this approach, a sequence of the HBV could be quantified over the range from 1.49x10(-7)M to 1.06x10(-6)M, with a linear correlation of r=0.9973 and a detection limit of 8.4x10(-8)M. The Cd(bzim)(2)(2+) signal observed from the probe sequence before and after hybridization with a four-base mismatch containing sequence was lower than that observed after hybridization with a complementary sequence, showing good selectivity. These results demonstrate that the Cd(bzim)(2)(2+) indicator provides great promise for the rapid and specific measurement of the target DNA.     

Mixed ligand complexes of osmium(II)-2,2-bipyridine: synthesis, spectral characterization and electrochemical properties of bis-chelated-arylazoimidazole-bipyridine-osmium(II) and X-ray crystal structure of [(2,2-bipyridine)-bis-{1-methyl-2-(p-tolylazo)imidazole}osmium(II) hexafluorophosphate

Reaction of ctc-OsBr₂(RaaiR^′)₂ [RaaiR^′=1-alkyl-2-(arylazo)imidazole, p-R-C₆H₄-NN-C₃H₂-NN-1-R^′, where R=H (a), Me (b), Cl (c) and R^′=Me (2), Et (3) and CH₂Ph (4)] with 2,2^′-bipyridine (bpy) in presence of AgNO₃ in EtOH followed by the addition of NH₄PF₆ afforded a mixed ligand complex [Os(bpy)(RaaiR^′)₂](PF₆)₂. The structure of the complex, in one case [Os(bpy)(MeaaiMe)₂](PF₆)₂ · 4H₂O, has been confirmed by X-ray crystallography. The complexes are diamagnetic (low spin d⁶, s=0) and they show intense MLCT transition in the visible region (480-525 nm) and a weak transition at longer wavelength (>850 nm) in CH₃CN solution. Cyclic voltammetry of the complexes show two metal oxidation, Os(II)/Os(III) at 0.72-0.76 V and Os(III)/Os(IV) at 1.34-1.42 V and three successive ligand reductions.     

Electrochemical behaviour and reactivity of [Os(bpy)2(CO)(OTf)] in halogenated solvents

The dimethylaminopyridine (DMAP) promoted reaction between [Os(bpy)₂(CO)(OTf)]OTf (where ) and methylene chloride is reported. C-Cl bond breaking of a solvent molecule leads to the formation of the [Os(bpy)₂(CO)(Cl)]OTf complex. The reactivity and redox properties of [Os(bpy)₂(CO)(OTf)]OTf were investigated by means of room- and low-temperature electrochemical experiments. In CH₂Cl₂, at low temperature, the complex undergoes two 1e electrochemical and chemical reversible reductions (E_rE_r mechanism), but at room temperature a more complex electrochemical mechanism is observed, leading to the electro-synthesis of [Os(bpy)₂(CO)(Cl)]OTf via electrochemical reversible and chemical irreversible reduction processes (E_rC_i mechanism). The DMAP nucleophilicity was used to produce the new [Os(bpy)₂(CO)(Br)]OTf and [Os(bpy)₂(CO)(I)]OTf complexes which have been fully characterized.     

Investigation of the interaction between ssDNA and 2-aminophenoxazine-3-one and development of an electrochemical DNA biosensor

An electrochemical method was used to probe the interaction between 2-aminophenoxazine-3-one (AP) and the short DNA sequence related to the hepatitis B virus (HBV), and an electrochemical DNA biosensor was developed. The voltammetric signals of AP have been investigated at bare glassy carbon electrode (bare GCE), hybrid double-stranded DNA-modified GCE (dsDNA/GCE), and single-stranded DNA-modified GCE (ssDNA/GCE) by means of differential pulse voltammetry (DPV), and the peak currents increased with respect to the order of electrodes. The extent of hybridization was evaluated on the basis of the difference between signals of AP with a probe before and after hybridization with the complementary sequence. Control experiments with noncomplementary were performed to test the selectivity of the biosensor. With this approach, a sequence of the HBV could be quantified over the range from 3.53 x 10(-7) to 1.08 x 10(-6) M, with a linear correlation of r = 0.9963 and a detection limit of 1.00 x 10(-7) M.     

Production and characterization of a polyclonal antibody for Os(II) and Ru(II) polypyridyl complexes

The characterization of a polyclonal antibody produced via immunization with an [Os(bpy)(2)dcbpy] hapten is described. Bpy is 2,2'-bipyridine and dcbpy is 2,2'-bipyridine-4,4'-dicarboxylic acid. The cross-reactivity of the antibody for the Ru(II) analogue of the hapten was also investigated. Large increases in the emission and luminescent lifetime of a series of Os and Ru complexes were observed on binding of the antibody. Association equilibrium constants were derived from luminescence titration data and were found to be 5.6 x 10(8) and 5.0 x 10(8)M(-1) for [Os(bpy)(2)dcbpy] and [Ru(bpy)(2)dcbpy], respectively. Spectroscopic changes were likely due to the exclusion of H(2)O from the complex/antibody binding cleft and blocking of vibrational relaxation pathways of the Os/Ru excited state. D(2)O/H(2)O experiments confirmed that the antibody protected approx. 82% of [Os(bpy)(2)dcbpy] and 80% of [Ru(bpy)(2)dcbpy] from excited state deactivation by the aqueous solvent.     

Electrogenerated chemiluminescence detection of adenosine based on triplex DNA biosensor

A novel electrogenerated chemiluminescence (ECL) biosensor based on the construction of triplex DNA for the detection of adenosine was designed. The ECL biosensor employs an aptamer as a molecular recognition element, and quenches ECL of tris(2,2'-bipyridine) ruthenium (Ru(bpy)(3)(2+)) by ferrocenemonocarboxylic acid (FcA). Through self-assembly technology, the ECL probe of thiolated hairpin adenosine aptamer tagged was self-assembled onto the surface of a gold electrode with an ECL signal producer Ru(bpy)(3)(2+) derivative (Ru-DNA-1). The adenosine aptamer, including a section of triplex characteristic chain, formatted triplex DNA with two other DNAs (DNA-2, Fc-DNA-3) in the presence of triplex DNA binder coralyne chloride (CORA). Fc-DNA-3 was tagged with an ECL quencher ferrocenemonocarboxylic acid (FcA), a quenching probe. In the presence of adenosine, the aptamer sequence (Ru-DNA-1) prefers to form the aptamer-adenosine complex with hairpin configuration and the switch of the DNA-1 occurs in conjunction with the generation of a strong ECL signal owing to the dissociation of a quenching probe. Meanwhile, a control experiment was performed; the ECL-duplex biosensor was designed to detect adenosine. The detection limits were 2.7×10(-10) mol L(-1) and 2.3×10(-9) mol L(-1) for the ECL-triplex DNA biosensor and ECL-duplex DNA biosensor, respectively, which demonstrated that the ECL-triplex DNA biosensor improved the sensitivity and specificity greatly.     

Synthesis,characterization and binding studies of chromium(III) complex containing an intercalating ligand with DNA

A chromium(III) complex [Cr(DPPZ)(2)Cl(2)](+), where DPPZ is a planar bidentate ligand with an extended aromatic system, has been found to bind strongly to CT DNA with an apparent binding constant of (1.8+/-0.5)x10(7) M(-1). The effects of [Cr(DPPZ)(2)Cl(2)](+) on the melting temperature and the viscosity of DNA clearly show that the chromium(III) complex interacts with DNA intercalatively. Competitive binding study shows that the enhancement in emission intensity of ethidium bromide (EthBr) in the presence of DNA was quenched by [Cr(DPPZ)(2)Cl(2)](+) indicating that the Cr(III) complex displaces EthBr from its binding site in DNA. The binding of this complex has been found to bring about B to Z conformational transition in CT DNA as well as poly(dG-dC).poly(dG-dC). Molecular modeling study also shows that binding energy of the complex with d(GC)(12) is much higher than Dickerson model and d(AT)(12). Modeling studies show that [Cr(DPPZ)(2)Cl(2)](+) brings about twist in the DNA base pairs as well as phosphate ester backbone resulting in conformational transition in DNA.     

Solute carrier family 26 member a2 (Slc26a2) protein functions as an electroneutral SOFormula/OH-/Cl- exchanger regulated by extracellular Cl-

Slc26a2 is a ubiquitously expressed SO(4)(2-) transporter with high expression levels in cartilage and several epithelia. Mutations in SLC26A2 are associated with diastrophic dysplasia. The mechanism by which Slc26a2 transports SO(4)(2-) and the ion gradients that mediate SO(4)(2-) uptake are poorly understood. We report here that Slc26a2 functions as an SO(4)(2-)/2OH(-), SO(4)(2-)/2Cl(-), and SO(4)(2-)/OH(-)/Cl(-) exchanger, depending on the Cl(-) and OH(-) gradients. At inward Cl(-) and outward pH gradients (high Cl(-)(o) and low pH(o)) Slc26a2 functions primarily as an SO(4)(2-)(o)/2OH(-)(i) exchanger. At low Cl(-)(o) and high pH(o) Slc26a2 functions increasingly as an SO(4)(2-)(o)/2Cl(-)(i) exchanger. The reverse is observed for SO(4)(2-)(i)/2OH(-)(o) and SO(4)(2-)(i)/2Cl(-)(o) exchange. Slc26a2 also exchanges Cl(-) for I(-), Br(-), and NO(3)(-) and Cl(-)(o) competes with SO(4)(2-) on the transport site. Interestingly, Slc26a2 is regulated by an extracellular anion site, required to activate SO(4)(2-)(i)/2OH(-)(o) exchange. Slc26a2 can transport oxalate in exchange for OH(-) and/or Cl(-) with properties similar to SO(4)(2-) transport. Modeling of the Slc26a2 transmembrane domain (TMD) structure identified a conserved extracellular sequence (367)GFXXP(371) between TMD7 and TMD8 close to the conserved Glu(417) in the permeation pathway. Mutation of Glu(417) eliminated transport by Slc26a2, whereas mutation of Phe(368) increased the affinity for SO(4)(2-)(o) 8-fold while reducing the affinity for Cl(-)(o) 2 fold, but without affecting regulation by Cl(-)(o). These findings clarify the mechanism of net SO(4)(2-) transport and describe a novel regulation of Slc26a2 by an extracellular anion binding site and should help in further understanding aberrant SLC26A2 function in diastrophic dysplasia.     

An integrated microfluidic system for rapid screening of alpha-fetoprotein-specific aptamers

An electrically neutral cobalt complex, Co(Eim)(4)(NCS)(2) (Eim=1-ethylimidazole, NCS=isothiocyanate) was synthesized and its interaction with double-stranded DNA (dsDNA) was comprehensively studied by electrochemical methods on a glassy carbon electrode (GCE). The experimental results revealed that the cobalt complex could interact with dsDNA via a specific groove-binding mode with an affinity constant of 3.6×10(5)M(-1). The surface-based studies showed that Co(Eim)(4)(NCS)(2) could electrochemically accumulate within the immobilized dsDNA layer rather than single-stranded DNA (ssDNA) layer. Based on this fact, the cobalt complex was utilized as an electrochemical hybridization indicator for the detection of oligonucleotides related to CaMV35S promoter gene. The results showed that the developed biosensor presented very low background interference due to the negligible affinity of the Co(Eim)(4)(NCS)(2) complex with ssDNA. The hybridization specificity experiments further indicated that the biosensor could well discriminate the complementary sequence from the base-mismatched and the non-complementary sequences. The complementary target sequence could be quantified over the range from 5.0×10(-9)M to 2.0×10(-6)M with a detection limit of 2.0×10(-10)M.     

Electrochemical DNA biosensor for the detection of interaction between di[azino-di(5,6-azafluorene)-kappa2-NN'] dibromicoppers and DNA

A new Cu(II) complex CuL(2)Br(2) (L = azino-di(5,6-azafluorene)-kappa(2)-NN') was synthesized, and a new method of electrochemical probe has been proposed for the determination of hepatitis B virus (HBV) based on its interaction with [CuL(2)](2+). This ligand, containing functional groups, as well as planar aromatic domains, is capable of binding to double-stranded DNA (dsDNA) more efficiently than to single-stranded DNA (ssDNA). Emphasis has been placed on the elucidation of the nature of the interaction by electrochemical techniques. The electroactive [CuL(2)](2+) could be employed as an electrochemical indicator to detect hybridization events in DNA biosensors. These biosensors have been constructed by immobilization of a probe DNA sequence from HBV onto glassy carbon electrode (GCE). After hybridization with the complementary target sequence, [CuL(2)](2+) was accumulated within the dsDNA layer. Electrochemical detection was performed by differential pulse voltammetry over the potential range. Using this approach, complementary target sequences of HBV can be quantified over the range of 1.74 x 10(-9) to 3.45 x 10(-7) M, with a detection limit of 8.32 x 10(-10) M and a linear correlation coefficient of 0.9936.In addition, this approach is capable of detecting hybridization of complementary sequences containing one or three mismatched bases.     

Using exonuclease III to enhance electrochemical detection of natural DNA damage in layered films

The natural double-stranded DNA (dsDNA) was immobilized on electrode surface by layer-by-layer assembly, forming PSS/PDDA/dsDNA films (PSS, poly(styrene-sulfonate); PDDA, poly(diallyldimethylammonium)), and used to detect DNA damage electrochemically. The DNA lesion induced by the alkylating agent methyl methanesulfonate (MMS) could be detected by cyclic voltammetry with ruthenium(II) tris(2,2'-bipyridyl) (Ru(bpy)(3)(2+)) in solution. After treated by E. coli exonuclease III enzyme, the electrocatalytic oxidation peak of the films was further amplified and greatly enhanced because the enzyme could convert those apurinic sites caused by MMS in the damaged dsDNA into single-stranded DNA regions and make more guanines in the DNA become exposed. This approach provided a novel idea for constructing DNA biosensor in sensitive screening of genetoxic chemicals in vitro.     

A signal-on electrochemiluminescence aptamer biosensor for the detection of ultratrace thrombin based on junction-probe

A novel signal-on junction-probe electrogenerated chemiluminescence (ECL) aptamer biosensor has been developed for the detection of ultratrace thrombin based on a structure-switching ECL-quenching mechanism. The ECL aptamer biosensor comprises two main parts: an ECL substrate and an ECL intensity switch. The ECL substrate was made by modifying the complex of Au nanoparticle and ruthenium (II) tris-bipyridine (Ru(bpy)(3)(2+)-AuNPs) on the surface of gold electrode (GE), and the ECL intensity switch contains three probes designed according to the "junction-probe" strategy. The first probe is capture probe (Cp) which was functionalized with a thiol group at one end and covalently attached to Ru(bpy)(3)(2+)-AuNPs modified GE through S-Au bonding. The second probe is aptamer probe (Ap), which containing 15-base anti-thrombin DNA aptamer. The third one is ferrocene-labeled probe (Fp), which was functionalized with ferrocene tag at one end. We demonstrated that, in the absence of thrombin, Cp, Ap and Fp will hybridize to form a ternary "Y" junction structure and resulted in a quenching of ECL of Ru(bpy)(3)(2+). Whereas, in the presence of thrombin, the Ap prefers to form the G-quadruplex aptamer-thrombin complex and lead to an obvious recovery of ECL of Ru(bpy)(3)(2+), which provided a sensing platform for the detection of thrombin. Using this reusable sensing platform, a simple, rapid and selective signal-on ECL aptamer biosensor for the detection of thrombin with a detection limit of 8.0×10(-15) M has been developed. The success in the present biosensor served as a significant step towards the development of monitoring ultratrace thrombin in clinical detection.     

The two modes of binding of Ru(phen)(2)dppz(2+) to DNA: thermodynamic evidence and kinetic studies

The binding of Ru(phen)(2)dppz(2+) (dppz=dipyrido[3,2-a:2',3'-c]phenazine) to DNA was investigated at pH 7.0 and 25 degrees C using stopped-flow and spectrophotometric methods. Equilibrium measurements show that two modes of binding, whose characteristics depend on the polymer to dye ratio (C(P)/C(D)), are operative. The binding mode occurring for values of C(P)/C(D) higher than 3 exhibits positive cooperativity, which is confirmed by kinetic experiments. The reaction parameters are K=2 x 10(3)M(-1), omega=550, n=1, k(r)=(1.9+/-0.5) x 10(7)M(-1)s(-1) and k(d)=(9.5+/-2.5)x10(3)s(-1) at I=0.012 M. The results are discussed in terms of prevailing surface interaction with DNA grooves accompanied by partial intercalation of the dppz residue. The other binding mode becomes operative for C(P)/C(D)<3 and the equilibria analysis shows this is an ordinary intercalation mode (K=1.3 x 10(6) M(-1), n=1.5 at I=0.012 M and K=2 x 10(5) M(-1), n=1.2 at I=0.21 M). Similar behaviour is displayed by double-stranded poly(A).     

Synthesis, crystal structure, magnetic property and oxidative DNA cleavage activity of an octanuclear copper(II) complex showing water-perchlorate helical network

A new octanuclear copper(II) complex has been synthesized and structurally characterized by X-ray crystallography: [Cu(8)(HL)(4)(OH)(4)(H(2)O)(2)(ClO(4))(2)].(ClO(4))(2).2H(2)O (1) (H(3)L=2,6-bis(hydroxyethyliminoethyl)-4-methyl phenol). The complex is formed by the linkage of two terminal bimetallic cationic units and a tetranuclear mu(3)-hydroxo bridged dicubane core by a very short intramolecular hydrogen bond (O-H...O, 1.48(3)A and the angle 175 degrees). The coordination sphere of the terminal copper atoms is square pyramidal, the apical positions being occupied by water and a perchlorate ion. Complex 1 self-assembles to form a new type of water-perchlorate helical network [(H(2)O)(2)(ClO(4))](infinity) involving oxygen atoms of coordinated perchlorate ion and the two lattice water molecules through hydrogen-bonding interaction. The variable temperature-dependent susceptibility measurement (2-300K) of 1 reveals a strong antiferromagnetic coupling, J(1)=-220cm(-1) and J(2)=-98cm(-1) (J(1) and J(2) representing the exchange constant within [Cu(2+)](4) and [Cu(2+)](2) units, respectively). The complex binds to double-stranded supercoiled plasmid DNA giving a K(app) value of 1.2x10(7)M(-1) and displays efficient oxidative cleavage of supercoiled DNA in the presence of H(2)O(2) following a hydroxyl radical pathway.     

A new photoelectrochemical biosensors based on DNA conformational changes and isothermal circular strand-displacement polymerization reaction

We report a strategy for the transduction of DNA hybridization into a readily detectable photoelectrochemical signal by means of a conformational change analogous to electrochemical DNA (E-DNA) approach. To demonstrate the effect of distance change for photosensitizer to the surface of electrode on the change of photocurrent, photosensitizer Ru(bpy)(2)(dcbpy)(2+) tagged DNA stem-loop structures were self-assembled onto a nanogold modified ITO electrode. Hybridization induced a large conformational change in DNA structure, which in turn significantly altered the electron-transfer tunneling distance between the electrode and photosensitizer. The resulting change in photocurrent was proportional to the concentration of DNA in the range of 1.0×10(-10)-8.0×10(-9)M. In order to improve the sensitivity of the photoelectrochemical biosensor, an amplified detection method based on isothermal strand displacement polymerization reaction was employed. With multiple rounds of isothermal strand replication, which led to strand displacement and constituted consecutive signal amplification, a detection limit of 9.4×10(-14)M target DNA was achieved.     

DNA intercalating studies of [Ru(bpy)2dmt]2+ with two vacant nitrogen atoms by introducing copper(II) ions

A novel, yet effective method for identifying DNA-binding modes of [Ru(bpy)(2)dmt](2+) (where bpy?=?2,2'-bipyridine and dmt?=?2,3-dimethyl-1,4,8,9-tetra-aza-triphenylene) on an indium tin oxide electrode has been successfully developed by introducing Cu(2+) ion and ethylenediaminetetraacetic acid. The results from emission spectra and fluorescence microscopic images suggested that [Ru(bpy)(2)dmt](2+) not only associates with Cu(2+) ion in both the absence and presence of DNA but also shows strong affinity with DNA in the presence of Cu(2+). Evidence for the strong binding of [Ru(bpy)(2)dmt](2+) to DNA was determined from the interface studies using electrochemical methods. The present study suggests that a combination of photoluminescence measurement with electrochemical methods identifies the DNA-binding behavior of luminescent molecules with redox activities. [Ru(bpy)(2)dmt](2+) binds to DNA via an intercalative mode.     

Synchronous fluorescence, UV-visible spectrophotometric, and voltammetric studies of the competitive interaction of bis(1,10-phenanthroline)copper(II) complex and neutral red with DNA

Constant wavelength synchronous fluorescence spectroscopy (CW-SFS), UV-visible absorption spectroscopy, and cyclic and differential pulse voltammetry were applied to investigate the competitive interaction of DNA with the bis(1,10-phenanthroline)copper(II) complex cation ([Cu(phen)(2)](2+)) and a fluorescence probe, neutral red dye (NR), in a tris-hydrogen chloride buffer (pH 7.4). The results show that both the [Cu(phen)(2)](2+)and the NR molecules can intercalate competitively into the DNA double-helix structure. The cyclic voltammetry method showed that both anodic and cathodic currents of [Cu(phen)(2)](2+) decreased on addition of the DNA and the intercalated [Cu(phen)(2)](2+)-DNA complex formed (beta = (4.14 +/- 0.24) x 10(3)). CW-SFS measurements were facilitated by the use of the three-way resolution of the CW-SFS for NR, [Cu(phen)(2)](2+), and NR-DNA. The important constant wavelength (CW) interval, Deltalambda, was shown to vary considerably when optimized (135, 58, and 98 nm for NR, NR-DNA, and [Cu(phen)(2)](2+), respectively). This approach clearly avoided the errors that otherwise would have arisen from the common assumption that Deltalambda is constant. Furthermore, a chemometrics approach, parallel factor analysis (PARAFAC), was applied to resolve the measured three-way CW-SFS data, and the results provided simultaneously the concentration information for the three reaction components, NR, [Cu(phen)(2)](2+), and NR-DNA, for the system at each equilibrium point. The PARAFAC analysis indicated that the intercalation of the [Cu(phen)(2)](2+) molecule into the DNA proceeds by exchanging with the NR probe and can be attributed to two parallel reactions. Comprehensive information was readily obtained; the replacement of the intercalated NR commenced immediately on introduction of [Cu(phen)(2)](2+), approximately 50% of NR was replaced by [Cu(phen)(2)](2+) at a concentration of 0.45 x 10(-5) mol L(-1), and nearly all of the NR was replaced at a [Cu(phen)(2)](2+) concentration of 2.50 x 10(-5) mol L(-1). This work has the potential to improve extraction of information from the fluorescence intercalator displacement (FID) assay.     

A novel alcohol dehydrogenase biosensor based on solid-state electrogenerated chemiluminescence by assembling dehydrogenase to Ru(bpy)(3)2+-Au nanoparticles aggregates

Based on electrogenerated chemiluminescence (ECL), a novel method for fabrication of alcohol dehydrogenase (ADH) biosensor by self-assembling ADH to Ru(bpy)(3)(2+)-AuNPs aggregates (Ru-AuNPs) on indium tin oxide (ITO) electrode surface has been developed. Positively charged Ru(bpy)(3)(2+) could be immobilized stably on the electrode surface with negatively charged AuNPs in the form of aggregate via electrostatic interaction. On the other hand, AuNPs are favourable candidates for the immobilization of enzymes because amine groups and cysteine residues in the enzymes are known to bind strongly with AuNPs. Moreover, AuNPs can act as tiny conduction centers to facilitate the transfer of electrons. Such biosensor combined enzymatic selectivity with the sensitivity of ECL detection for quantification of enzyme substrate, and it displayed wide linear range, high sensitivity and good stability.