Electrochemical detection of beta-1,3-glucanase gene from transgenic capsicums using asymmetric PCR generated by a detecting probe and an anchoring probe.

A design for recognition of beta-1,3-glucanase gene (Glu) specific sequence based on probe extension was described. The detecting probe DNA and the anchoring prober were hybridized with the same target DNA firstly, then the probes were extended by DNA polymerase reaction. After that the double strand DNA was denatured, and the extended detecting probe was immobilized on a glassy carbon electrode via nanoparticle gold (AuNP). In electrochemical detection (cyclic voltammetry, CV and differential pulse voltammetry, DPV), an increased peak current (i(p)) of the indicator (methylene blue, MB) was obtained compared with the probe without extension. Three differently long DNAs of Glu specific sequence were employed as the target: oligonucleotide acid, molecular cloning vector DNA and total genome DNA of transgenic capsicum. The estimated DPV detection limits for three targets of oligonucleotide, the molecular cloning vector DNA and genome DNA were 2.6x10(-13), 6.0x10(-13) and 8.0x10(-13)moll(-1) respectively.

An electrochemical assay of beta-1,3-glucanase gene from transgenic capsicum using asymmetric PCR.

5'-Thiol-derivatized specific DNA probes were added to the single primer polymerase chain reaction (asymmetric PCR) solution. In the PCR process, the DNA probes extended in the presence of target; the extended probes were then immobilized on a glassy carbon electrode (GCE) via gold nanoparticles. Finally, methylene blue and the extended probes were combined and the electrochemical signals were measured. This signal was higher than that of the GCE modified only by the original probe. When there was no target in PCR solution, the probe did not extend and the signal did not increase. The specific sequences of the beta-1,3-glucanase gene were detected successfully from three targets with different length: oligonucleotide, molecule clone vector DNA, and total genome DNA of transgenic capsicum. The detection limits of 2.6 x 10(-13), 7.8 x 10(-13), and 9.1 x 10(-13) moll(-1) for oligonucleotide, molecule clone vector DNA, and total transgenic capsicum genome DNA were estimated.

A new scheme of hybridization based on the Au(nano)-DNA modified glassy carbon electrode.

DNA hybridization on the Au(nano)-DNA modified glassy carbon electrode (GCE) was investigated. The thiol modified probe oligonucleotides (SH-ssDNA) at the 5' phosphate end were assembled on the Au(nano)-DNA modified GCE surface. The electrochemical response of the probe immobilization and hybridization with target DNA was measured by differential pulse voltammetry (DPV) using methylene blue (MB) as the electroactive indicator. Gold nanoparticles can be dispersed effectively on the GCE surface in the presence of calf thymus DNA. Au(nano)-DNA modified GCE could greatly increase the active sites and enhance the response signal during immobilization and hybridization. The hybridization amount of target DNA could be greatly increased. The linear detection range of Au(nano)-DNA electrode for the complementary 21-mer oligonucleotide (cDNA) was achieved from 1.52 x 10(-10) to 4.05 x 10(-8) mol L(-1). The detection limit could reach the concentration of 10(-10) mol/L.

Electrochemical study on behavior of EuMo2 complex and its interaction with DNA.

The electrochemical behavior of complex EuMo2 (Mo = Morin, 2',3,4'5,7-pentahydroxyflavone) and its interactions with calf thymus DNA were studied using cyclic voltammetry (CV) and double potential step chronocoulometry (DPSCC) at glass carbon electrode (GCE) and DNA modified GCE, respectively. Information such as diffusion coefficient (D), rate constant (ks) of EuMo2 and intrinsic binding constant (K), binding numbers (n) of bound species per DNA (bp) were obtained. EuMo2 can bind to DNA, and the binding mode is intercalation. By nonlinear fitting with Langmuir equation, a K of 1.02 x 10(6) M-1 and an n of 1 were obtained.

Electrochemical detection of scDNA cleavage in the presence of macrocyclic hexaaza-copper(II) complex.

The hexaaza macrocyclic copper(II) complex Cu(II)L(L=1,8-Dihydroxyethyl-1,3,6,8,10,13-hexaazacyclotetradecane), which has octahedral structure similar to some natural complexes, is synthesized and purified. In this study, oxidative breakage DNA by the reaction of Cu(II)L with H2O2 and ascorbate has been investigated by gel electrophoresis experiments. In electrochemical experiments, the on scDNA-modified glassy carbon electrode(GCE) is cleaved by the Cu(II)L and redox changing of the metal catalyst without adding any other reagents. Above all, the need for concentration of scDNA is much lower than that of gel electrophoresis experiments and the process of the performance is easy. Furthermore, Cyclic Voltammetry (CV) and A.C. Impedance, which are performed to monitor scDNA cleavage at the scDNA-modified glassy carbon electrode (GCE), are fast, simple and highly efficient. The mechanism of the damage can be suggested: Fenton.

Electrochemical study on the behavior of Morin and its interaction with DNA.

Voltammetric behavior of Morin was studied in 0.1M HAc-NaAc+50mM KCl (pH 3.4) solution at glassy carbon electrode (GCE) using cyclic voltammetry (CV). Morin showed an irreversible anodic peak at 0.720 V in CV which was involving two electrons and two protons. Also, the interaction of Morin with double-stranded calf thymus DNA (ctDNA) was studied by CV at GCE with an irreversible electrochemical equation. As a result of reaction with ctDNA, the voltammetric peak of Morin was a position shift and the peak current decreased. The diffusion coefficients of both free and binding Morin (D(f)=1.1,086 x 10(-7)cm(2)s(-1) and D(b)=8.2,544 x 10(-9)cm(2)s(-1)), binding constant (K=1.7,765 x 10(7)cm(3)mol(-1)), and binding site size (s=0.8,510) of the Morin-DNA complex were obtained simultaneously by non-linear fit analysis. The results demonstrate that Morin can bind to ctDNA in 0.1M HAc-NaAc+50mM KCl (pH 3.4) solution and the ring B of Morin intercalates between the DNA base pairs.

Mechanism of DNA Strand Cleavage Induced by Hexaaza Macrocyclic Nickel (II) Complex.

The hexaaza macrocyclic nickel(II) complex (Ni(II)L-1,8-Dihydroxyethyl-1,3,6,8,10,13-hexaazacyclotetradecane nickle(II) perchlorate monohydrate) was synthesized and purified. The electrochemical character of Ni(II)L was measured, and the interaction of Ni(II)L with calf thymus DNA (CT-DNA) was studied using electrochemical techniques, emission and viscometry, and circular dichroic spectral measurements. All of the experiments suggested that the complex interacted with DNA primarily by partial intercalation. The cyclic voltammetry (CV) showed that the currents of both the reduction peak and the oxidation peak decreased significantly in the presence of DNA, which indicated that Ni(II)L could interact with DNA. The fluorescence intensity of the DNA-ethidium bromide(EB) system decreased distinctly when Ni(II)L was added. The results indicated that Ni(II)L may be completed effectively with EB for the intercalative binding sites. The viscosity of DNA would be decreased slightly by the addition of the complex. Circular dichroic spectral studies revealed that B conformation of CT-DNA became more A-like in structure on interaction with the complex. Noticeably, the complex has been found to cleave plasmid pBR 322 by agarose gel electrophoresis and cleave CT-DNA by high-performance liquid chromatography (HPLC).

Study on the interaction of new water-soluble porphyrin with DNA.

A porphyrin meso-tetrakis{[4-(1-pyridyl)propoxy]phenyl}porphyrin (TPyPP) and its Ni complex (TPyPP(Ni)) have been synthesized and characterized by 1H NMR, UV-vis spectra. The interaction of two porphyrins with calf thymus-DNA (CT-DNA) has been explored by UV-vis, fluorescence and circular dichroic spectroscopy and viscosity measurements. The results suggest that these porphyrins can bind to DNA by the same binding mode. TPyPP outside binds by self-stack with DNA both at low drug load r (=[porphyrin]/[DNA]) and high drug load. Though TPyPP(Ni) has center metal nickel, binding mode with DNA has little difference compared with TPyPP, dominating out-binding mode with different direction along DNA. The binding constants of the TPyPP and TPyPP(Ni) to DNA were 4.65 x 10(5) M(-1) and 3.2 x 10(5) M(-1), respectively. A colored precipitate was found after time in two porphyrin's viscosity measurement. The reasonable interpretation is the porphyrins with alkyl connected N-position of pyridine can strongly interact with the anionic phosphates of DNA and lead to hydrophobic complex.

Electrochemical studies of kanamycin immobilization on self-assembled monolayer and interaction with DNA.

The electrochemical characteristics of kanamycin onto self-assembled monolayer (SAM) modified gold electrode (SAM/Au) is investigated by cyclic voltammetry. In the potential range 0-0.6 V, Cu(II) yields a pair of stable redox waves at the bare gold electrode. E(pa) is located at 0.189 V and E(pc) at 0.254 V. In contrast, Cu(II) is reduced at a more positive potential and a decreasing current at the kanamycin SAM/Au electrode. Cu(II) and kanamycin can form a stoichiometry complex with chemical ratio of 2:1. The interaction of Cu(II)-kanamycin complex with calf thymus DNA is also studied in the solution. And the interactive mode between Cu(II)-kanamycin complex and DNA is verified by the fluorescence method. Binding constants (K) of the Cu(II)-kanamycin complex to DNA and binding site size (s) are calculated from voltammetric data and equal to 1.5 x 10(7) l/mol and 4 bp, respectively.

Electrochemical investigation on interaction between DNA with quercetin and Eu-Qu3 complex.

The interactions of quercetin (Qu) and Eu-Qu3 complex with calf thymus DNA were studied using cyclic voltammetry (CV) and double potential step chronocoulometry (DPSCC) at glass carbon electrode (GCE) for the surface method. The method is simple, convenient, reliable, reagent saving. Information such as intrinsic binding constant (K), and binding numbers (n) of bound species per DNA (bp), ratio (K(Ox)/K(Red)) of the binding constants for the oxidized and reduced forms of a bound species and interaction mode was obtained using dsDNA-modified GCE. Quercetin and Eu-Qu3 can both bind to DNA, but quercetin binds to DNA mainly by electrostatic attraction and the complex bind to DNA by both intercalation and electrostatic attraction. For the quercetin/dsDNA-modified GCE systems, a K of (3.80+/-0.3) x 10(4) M(-1), saturation coverage value (Gammas) of (2.28+/-0.2) x 10(-10) mol/cm2 and n of 1.2 were obtained. For the complex system, a saturation coverage value (Gammas) of 1.65 x 10(-10) mol/cm2 and n of 1.8 were obtained.

[Comparative study of room-temperature phosphorescence of 1-bromonaphthalene induced by synergetic effect of nonionic surfactants and beta-cyclodextrin].

Room-temperature phosphorescence of 1-BrN induced by a combination of OPE-10 and Triton X-100 with beta-CD was comparatively studied. In terms of molecular size and dimensions of beta-CD, the octyl group and phenyl group of OPE-10 and Triton X-100 were incorporated into the cavity of beta-CD and the complexes with the stoichiometry of 1:1 were formed. The removal of water molecules inside the cavity results in a greater apolar interior. By enhanced hydrophobic interaction, the cavity occupied by OPE-10 and Triton X-100 is able to further capture another 1-BrN and form close packing 1:1:1 ternary inclusion complexes with apparent stability constant of 1.09 x 10(5) and 4.47 x 10(5) L2 x mol(-2), respectively. 1-BrN shows bright phosphorescence at room temperature due to the greater rigidity in the limited space and the favorable microenvironment shielding from external quenchers and quenching effect on the fluorescence of the phenyl group of OPE-10 and Triton X-100 within the same cavity. In the case of Triton X-100, the larger tert-octyl group better shields off external quenchers such as dissolved oxygen and iodide ion. Energy transfer from the excited phenyl group of Triton X-100 to adjacent 1-BrN acceptor was observed.

Comparative study on fluorescence enhancement and quenching of europium and terbium chelate anions in cationic micelles.

Fluorescence enhancement and quenching of water soluble chelates of terbium (Tb3+) with Tiron, salicylic acid (SA), 4-sulfonyl salicylic acid (SSA) and acetylacetone (AA) and sparingly soluble chelates of europium (Eu3+) with beta-diketones were comparatively examined in the presence of cetyltrimethyl ammonium bromide (CTMAB) and cetylpyridinium chloride (CPC). By the composition of the complexes, surface tension measurements and spectral analysis, the binding mode of chelate anions to the micellar surface of cationic surfactants was discussed in terms of ion-exchange model. Quenching effect of CPC on the fluorescence of association complexes seems to arise from the charge transfer from a fluorescent ligand to pyridinium cation. In the case of the chelates of Eu3+ with beta-diketones, however, pyridinium ion is only capable of overlapping the aromatic ring of beta-diketones to less extent since the poorly soluble charged chelates have a weak affinity for the highly polar surface of pyridinium cationic micelles. Efficient charge transfer between the excited aromatic beta-diketone and pyridinium cation fails to be established. CPC also shows enhanced effect on fluorescence like CTMAB.

Plasma degradation of dyes in water with contact glow discharge electrolysis.

Contact glow discharge electrolysis (CGDE) of two dyes, weak acid brilliant red B and weak acid flavine G, was investigated under different concentrations, temperature and mediums. From the variation of their concentration with the reaction time, it was demonstrated that the oxidation would be a first-order reaction. On the base line of UV spectra of solution in the degradation process, we deduced that two dyes underwent the oxidative degradation in CGDE. The rate constants, relevant coefficients and the decolorization degree were displayed under different conditions.

Voltammetric studies of the interaction of transition-metal complexes with DNA.

The interaction of the two new synthesized transition-metal complexes, ML(2) (M=Co, Cu, L=1,8-dihydroxyethyl-1, 3,8,10,13-hexa-azacyclotetradecane) with calf thymus DNA was probed by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). Adding deoxyribonucleic acid (DNA) into [CoL](2+) and [CuL](2+) solution, the i(p) value of all the peaks of [CoL](2+) and [CuL](2+) significantly decreased in proportion to concentration of DNA. Glassy carbon electrodes (GCEs) were modified with DNA by adsorption, and it was electrochemically characterized with transition-metal complexes, [ML](2+). The DNA modification layer on the GCE is unstable to alkali and to heat, but stable to acid solutions and very stable in long stock in a dry state. It could be seen that peak potential shifted positively and the peak current increased significantly. The electrochemical parameters, binding constant (k(n+)) and binding sites(s) were calculated by a nonlinear regression method.

Study on the interactions between CuL(2) and Morin with DNA.

Absorption, fluorescence spectral and viscometric studies have been carried out on the interaction of Morin (2',3,4',5,7-pentahydroxyflavone, ) and its Cu complex, CuL(2) x 2H(2)O [L=Morin (2'-OH group deprotonated), ] with calf thymus DNA. CuL(2) shows different spectral characteristics from that of Morin in the presence of DNA. Increasing fluorescence is seen for CuL(2) with DNA addition whereas decreased fluorescence is observed for Morin. Quenching fluorescence is observed for the DNA-EB system when CuL(2) is added whereas slightly quenched fluorescence is seen for the DNA-EB system with Morin addition. The relative viscosity of DNA and the DNA-EB system increases with the addition of CuL(2.) Hypochromism and a smaller shift are observed in the UV-visible spectra of CuL(2) in the presence of DNA and the denatured temperature of DNA is decreased in the presence of CuL(2). The above results suggested that Morin and CuL(2) can both bind to DNA, but the binding mode is different. The complex binds to DNA mainly by intercalation, while Morin binds in a nonintercalating mode.

Determination of glutamic acid by an oscillating chemical reaction using the analyte pulse perturbation technique.

An analytical method for the determination of glutamic acid by the sequential perturbation caused by different amounts of glutamic acid on the oscillating chemical system involving the Cu(II)-catalyzed oscillating reaction between hydrogen peroxide and sodium thiocyanate in an alkaline medium is proposed. The method relies on the linear relationship between the changes in the oscillation amplitude of the chemical system and the concentration of glutamic acid. The reaction is implemented in a continuous-flow stirred-tank reactor, and changes in the oscillation amplitude on each perturbation are proportional to the glutamic acid concentration. The use of the analyte pulse perturbation (APP) technique permits sequential determinations on the same oscillating system owing to the expeditiousness with which the steady state is regained after each perturbation. The dynamic range lies between 2.5x10(-6) and 3.2x10(-4) M of glutamic acid, with the regression coefficient is 0.9987. The precision is excellent (less than 0.68% as relative standard deviation (R.S.D.)). Some aspects of the potential mechanism of action of glutamic acid on the oscillating system are discussed.