The Analysis of Cu(II)/Zn(II) Cyclopeptide System as Potential Cu,ZnSOD Mimic Center

In this paper are presented the features of copper (II) and zinc (II) heteronuclear complexes of the cyclic peptide—c(HKHGPG)₂. The coordination properties of ligand were studied by potentiometric, UV–Vis and CD spectroscopic methods. These experiments were carried out in aqueous solutions at 298 K depending on pH. It turned out that in a physiological pH dominates Cu(II)/Zn(II) complex ([CuZnL]⁴⁺) which could mimic the active center of superoxide dismutase (Cu,ZnSOD). In next step we performed in vitro research on Cu,ZnSOD activity for [CuZnL]⁴⁺ complex existing in 7.4 pH by the method of reduction of nitroblue tetrazolium (NBT). Also mono- and di-nuclear copper (II) complexes of this ligand were examined. The ability of inhibition free radical reaction were compared for all complexes. The results of these studies show that Cu(II) mono-, di-nuclear and Cu(II)/Zn(II) complexes becoming to new promising synthetic superoxide dismutase mimetics, and should be considered for further biological assays.     

黄曲霉毒素氧化酶/壳聚糖-单壁碳纳米管/聚邻苯二胺修饰电极对杂色曲霉素的检测

将黄曲霉毒素氧化酶(AFO)固定在壳聚糖(CS)-单壁碳纳米管(SWCNTs)杂交膜中,组装在聚邻苯二胺(POPD)修饰的金电极(Au)表面,制备了对杂色曲霉素(ST)敏感的生物传感器(AFO/CS-SWCNTs/POPD/Au)。运用原子力显微镜(AFM)、傅立叶变换红外光谱(FT-IR)和交流阻抗技术(EIS)对电极组装过程进行了表征。循环伏安法研究表明,AFO在修饰电极上发生了准可逆的氧化还原反应,是表面控制过程,其式量电位为-0.436V(vs.Ag/AgCl),说明包埋在CS-SWCNTs中的AFO和电极之间发生了直接电子传递。AFO修饰电极对ST具有明显的电催化作用,其表观米氏常数appKM为7.13μmol/L,催化电流与ST浓度在10～310ng/mL范围内呈线性关系,相关系数为0.997,检出限为3ng/mL(S/N=3),响应时间小于10s。组装的生物传感器具有较好的稳定性与重现性,连续检测20ng/mL的ST标准溶液11次,电流值RSD为3.9%;放置一个月后,其电流响应值仍为初始值的85.6%。该方法具有较高的选择性和灵敏度,应用于实际样品检测时,其回收率在87.6%～105.5%之间...     

Speciation study of an imidazolate-bridged copper(II)-zinc(II) complex in aqueous solution

The solution equilibrium and the binding mode of the species in the five-component system containing two metal ions (copper(II) and zinc(II)) and three ligands (A=diethylenetriamine, B=imidazole, C=tris(2-aminoethyl)amine) were investigated by pH-potentiometric titration, UV-visible spectrophotometry and EPR (electron paramagnetic resonance) spectroscopic titration in aqueous solution in the 2-11 pH range. An imidazolate-bridged heterobinuclear complex (ACuBH(-1)ZnC) was found to evolve above pH=7 and was stable between pH 7 and 11. The existence of the ACuBH(-1)ZnC complex (by determination of its molecular weight) was proved by mass spectrometry (ESI-MS (electrospray ionization mass spectrometry) and MALDI (matrix-assisted laser desorption/ionization) techniques). The electrochemical behaviour and the superoxide dismutase activity of this complex were also tested by cyclic voltammetry and the Riboflavin/NBT (nitro blue tetrazolium) assay, respectively.     

Cobalt(II) salophen-modified carbon-paste electrode for potentiometric and voltammetric determination of cysteine

A chemically modified electrode constructed by incorporating N,N(')-bis(salicylidene)-1,2-phenylenediaminocobalt(II) into carbon-paste matrix was used as a sensitive electrochemical sensor for detection of cysteine. The resulting electrode exhibits catalytic properties for the electrooxidation of cysteine and lowers the overpotential for the oxidation of this compound. The faster rate of electron transfer results in a near-Nernstian behavior of the modified electrode and makes it a suitable potentiometric and voltammetric sensor for the fast and easy determination of cysteine. A linear response in concentration range from approximately 2 microM to 0.01 M was obtained with a detection limit of 1 microM for the potentiometric detection of cysteine. The modified electrode was also used for the amperometric and differential pulse voltammetric determination of cysteine and the results were compared with those of the potentiometric method.     

Hetero-association of caffeine and aromatic drugs and their competitive binding with a DNA oligomer

NMR spectroscopy has been used to elucidate the molecular basis of the action of caffeine (CAF) on the complexation with DNA of mutagens such as ethidium bromide, propidium iodide, proflavine and acridine orange, and anticancer drugs such as actinomycin D and daunomycin. The hetero-association of CAF and each of the aromatic ligands in 0.1 mol L(-1) phosphate buffer (pD=7.1) has been investigated as a function of concentration and temperature by 500 MHz 1H NMR spectroscopy and analysed in terms of a statistical-thermodynamic model, in which molecules form indefinite aggregates for both self-association and hetero-association. The analysis leads to determination of the equilibrium constants of hetero-association and to the values of the limiting chemical shifts of the heteroassociation of CAF with each of the aromatic molecules. The hetero-association constants between CAF and each of the aromatic drugs/dyes are found to be intermediate in magnitude between those for self-association of CAF and the corresponding drug/dye. The most probable structures of the 1:1 CAF + ligand hetero-association complexes have been determined from the calculated values of the induced limiting chemical shifts of the drug protons. Knowledge of the equilibrium constants for self-association of CAF and the aromatic ligands, for their hetero-association and their complexation with a DNA fragment, the deoxytetranucleotide 5'-d(TpGpCpA), enabled the relative content of each of the CAF-ligand and CAF-ligand-d(TGCA) complexes to be calculated as a function of CAF concentration in mixed solutions. It is concluded that, on addition of CAF to the solution, the decrease in binding of drug or mutagen with DNA is due both to competition for the binding sites by CAF and the aromatic molecules, and to formation of CAF-ligand hetero-association complexes in the mixed solution; the relative importance of each process depends on the drug or mutagen being considered.     

De-intercalation of ethidium bromide and propidium iodine from DNA in the presence of caffeine

Caffeine (CAF) is capable of interacting directly with several genotoxic aromatic ligands by stacking aggregation. Formation of such hetero-complexes may diminish pharmacological activity of these ligands, which is often related to its direct interaction with DNA. To check these interactions we performed three independent series of spectroscopic titrations for each ligand (ethidium bromide, EB, and propidium iodine, PI) according to the following setup: DNA with ligand, ligand with CAF and DNA-ligand mixture with CAF. We analyzed DNA-ligand and ligand-CAF mixtures numerically using well known models: McGhee-von Hippel model for ligand-DNA interactions and thermodynamic-statistical model of mixed association of caffeine with aromatic ligands developed by Zdunek et al. (2000). Based on these models we calculated association constants and concentrations of mixture components using a novel method developed here. Results are in good agreement with parameters calculated in separate experiments and demonstrate de-intercalation of EB and PI molecules from DNA caused by CAF.     

Direct electrochemistry and electrocatalysis of heme proteins immobilized on gold nanoparticles stabilized by chitosan

Three heme proteins, myoglobin, hemoglobin, and cytochrome c, have been adsorbed onto chitosan-stabilized gold nanoparticles (Chit-Aus) modified Au electrode via a molecule bridge like cysteine. UV-vis spectra indicated that the proteins on Chit-Aus films retained near-native secondary structures. The fabricated procedures and electrochemical behaviors of proteins on such an interface were characterized with electrochemical impedance spectra and cyclic voltammetric techniques. It was demonstrated that Chit-Aus film could not only offer a friendly environment to immobilize protein molecules but also enhance the electron transfer ability between protein molecules and underlying electrode. The effects of scan rate and pH on the electrochemical behaviors of each heme protein are discussed in detail. The resultant electrode displayed an excellent electrocatalytic response to the reduction of H(2)O(2), long-term stability, and good reproducibility.     

Synthesis and Cu(II) coordination of two new hexaamines containing alternated propylenic and ethylenic chains: Kinetic studies on pH-driven metal ion slippage movements

The synthesis of the open-chain and cyclic polyamines, 1,5,8,12,15,19-hexaazaheptadecane (L1) and 2,6,9,13,16,20-hexaaza[21]-(2,6)-pyridinophane (L2), are described. The protonation constants and interaction constants with Cu(II) have been determined by potentiometric measurements carried out at 298.1 K in 0.15 mol dm⁻³ NaClO₄. The values obtained are discussed as a function of the open-chain or cyclic nature of the ligands and compared with analogous polyamines containing different sets of hydrocarbon chains between the nitrogen donors. Kinetic studies on the acid-promoted dissociation of the Cu(II) complexes indicate that the mono and binuclear complexes of L1 decompose with different kinetics, a behavior unprecedented for open-chain polyamines. In contrast, the dissociation of the first metal ion is accelerated in the binuclear complexes of L2 and so, all the mono and binuclear complexes of L2 decompose with the same kinetics. The voltammetric response of Cu(II)-L1 and Cu(II)-L2 complexes has been studied in order to correlate electrochemical and structural data.     

Neurotransmitter dopamine applied in electrochemical determination of aluminum in drinking waters and biological samples.

It was demonstrated that the decrease of the differential pulse voltammetric (DPV) anodic peak current of dopamine (3,4-dihydroxyphenylethylamine, DA) was linear with the increase of aluminum (Al) concentration. Under optimum experimental conditions (pH 4.6, 1.2 x 10(-3) M DA, and 0.04 M NaAc-HAc buffer solution), the linear range is 4.0 x 10(-7)-8.0 x 10(-5) M, the detection limit is 1.4 x 10(-7) M, and the relative standard deviation for 4 x 10(-5) M Al(III) is 3.5% (n=8). Many foreign species, especially some low-molecule-weight biological molecules, were chosen for interference testing. The proposed method was applied to the determination of Al in biological samples such as synthetic renal dialysate, Ringer's solution, human blood, cerebrospinal fluid of a patient, and urine of a diabetic patient. The corresponding recoveries were generally between 95 and 105%. The basic principle of the method was determined by examining Al complexed with DA. This results in the blockage of the electroactive sites on DA, followed eventually by the reduction of the electrochemical response of DA. This result was verified by examining the behavior of DA, both in the presence and absence of Al, using electrochemical, UV-Vis, Raman, and (13)C NMR spectroscopic methods.     

Deep oxidation of glucose in enzymatic fuel cells through a synthetic enzymatic pathway containing a cascade of two thermostable dehydrogenases

A sensitive glutamate biosensor is prepared based on glutamate dehydrogenase/vertically aligned carbon nanotubes (GLDH, VACNTs). Vertically aligned carbon nanotubes were grown on a silicon substrate by direct current plasma enhanced chemical vapor deposition (DC-PECVD) method. The electrochemical behavior of the synthesized VACNTs was investigated by cyclic voltammetry and electrochemical impedance spectroscopic methods. Glutamate dehydrogenase covalently attached on tip of VACNTs. The electrochemical performance of the electrode for detection of glutamate was investigated by cyclic and differential pulse voltammetry. Differential pulse voltammetric determinations of glutamate are performed in mediator-less condition and also, in the presence of 1 and 5 μM thionine as electron mediator. The linear calibration curve of the concentration of glutamate versus peak current is investigated in a wide range of 0.1-500 μM. The mediator-less biosensor has a low detection limit of 57 nM and two linear ranges of 0.1-20 μM with a sensitivity of 0.976 mA mM(-1) cm(-2) and 20-300 μM with a sensitivity of 0.182 mA mM(-1) cm(-2). In the presence of 1 μM thionine as an electron mediator, the prepared biosensor shows a low detection limit of 68 nM and two linear ranges of 0.1-20 with a calibration sensitivity of 1.17 mA mM(-1) cm(-2) and 20-500 μM with a sensitivity of 0.153 mA mM(-1) cm(-2). The effects of the other biological compounds on the voltammetric behavior of the prepared biosensor and its response stability are investigated. The results are demonstrated that the GLDH/VACNTs electrode even without electron mediator is a suitable basic electrode for detection of glutamate.     

Nano-composition of riboflavin-nafion functional film and its application in biosensing

A novel nafion-riboflavin membrane was constructed and characterized by the scanning electron microscopy (SEM), transmission electron microscopy (TEM), UV-visible spectroscopy and cyclic voltammetric techniques. The estimated average diameter of the designed nanoparticles was about 60 nm. The functional membrane showed a quasi-reversible electrochemical behaviour with a formal potential of -562 +/- 5 mV (vs Ag/AgCl) on the gold electrode. Some electrochemical parameters were estimated, indicating that the system has good and stable electron transfer properties. Moreover, horseradish peroxidase (HRP) was immobilized on the riboflavin-nafion functional membrane. The electrochemical behaviour of HRP was quasi-reversible with a formal potential of 80 +/- 5 mV (vs Ag/AgCl). The HRP in the film exhibited good catalytic activity towards the reduction of H2O2. It shows a linear dependence of its cathodic peak current on the concentration of H2O2, ranging from 10 to 300 (micro)M.     

Synthesis, spectroscopic, structural and electrochemical studies of carboxyl substituted 1,4-naphthoquinones

Two carboxyl substituted quinones and their ethyl esters were prepared by alkylation of 2-methyl-1,4-naphthoquinone (MNQ), also known as menadione or vitamin K₃. All products were characterized by spectroscopic (¹H NMR, ¹³C NMR, IR) and electrochemical (cyclic voltammetry) methods, and the crystal structure of the two carboxylic derivatives was also determined. Both carboxyl substituted quinones crystallize in the system as hydrogen bonded dimers. In MeCN, the cyclic voltammograms of the ester derivatives present two reversible one-electron redox waves very similar to those of the parent quinone, MNQ. However, in the same solvent, the corresponding carboxyl substituted quinones show one cathodic and one anodic additional irreversible waves at more positive potentials and a decrease in current intensity of the two quinone reduction waves accompanied by loss of the quasi-reversible character of the second wave. These results show that the presence of the carboxylic substituent does not greatly modify the redox behaviour of the quinone, except for a small anodic shift of the potentials, but the associated presence of H⁺ ions in solution causes an important perturbation to the system, stabilizing the electrogenerated semiquinones by intermolecular self-protonation and/or hydrogen bonding.     

Superoxide-dependent and superoxide-independent pathways for reduction of nitroblue tetrazolium in isolated rat cardiac myocytes.

Spectroscopic studies indicated that nitroblue tetrazolium (NBT) could be reduced to blue formazan by several distinct reactions in suspensions of isolated rat cardiac myocytes. Both NADPH- and NADH-linked pathways for reduction of NBT were observed. NADPH-linked NBT reduction showed little activity in the absence of digitonin, but could be stimulated an average of 9.5-fold by digitonin permeabilization of the plasma membrane. NADH-linked NBT reduction occurred in the absence of digitonin, and could be increased an average of 3.5-fold by digitonin treatment. Analysis of the effects of cell viability on the extent of digitonin stimulation with these substrates suggested that the NADPH-linked reaction involved a cytosolic component, while the NADH-linked reaction involved an intracellular membrane enzyme system. With either NADPH or NADH, NBT reduction was completely inhibited by dicoumarol (100 microM). Dicoumarol-insensitive NBT reduction could subsequently be observed following the addition of 2 mM cyanide, a level of cyanide known to inhibit cytosolic superoxide dismutase. Cyanide-stimulated, dicoumarol-insensitive NBT reduction was augmented by the presence of either antimycin or doxorubicin, two agents which enhance superoxide formation by different mechanisms. The results indicate the existence of multiple pathways for both superoxide-independent and superoxide-dependent reduction of NBT. Dicoumarol-insensitive, cyanide-stimulated NBT reduction may be useful as a spectroscopic probe for intracellular superoxide formation.     

Copper-psychoactive drug complexes: a voltammetric approach to complexation by 1,4-benzodiazepines

Copper complexation by the 1,4-benzodiazepines medazepam, diazepam, flurazepam, nitrazepam, and clonazepam was investigated using differential pulse polarography and cyclic voltammetry at a mercury electrode in 0.10 M KNO3 and pH 7.0 +/- 0.1. Because the 1,4-benzodiazepines are easily reduced at a mercury electrode through the two-electron reduction of the 4,5-azomethine functional group, copper reduction, as well as that of the ligands, was analyzed under varying experimental conditions. In most situations adsorption phenomena occurred and their influence on voltammetric signals had to be carefully analyzed. The voltammetric behavior was then interpreted in terms of complex formation. The results showed that all benzodiazepines can act as ligands toward copper(II) ions, forming 1:1 and 1:2 complexes with similar stabilities. The stoichiometric acidity constants of the benzodiazepines under study were also determined by potentiometric titration in water-ethanol medium and 0.10 M KNO3 and then extrapolated to 0% concentration of ethanol.     

Ultrasensitive electrochemical immunosensing using magnetic beads and gold nanocatalysts

In the current study, we developed a nanocatalyst-based electrochemical immunoassay using magnetic beads (MBs) and gold nanocatalysts (AuNs). The MBs conjugated with IgG allow easy separation of target proteins and rapid immunosensing reaction, and the AuNs conjugated with IgG amplifies electroactive species via catalytic reaction of AuNs. An antimouse IgG-MB conjugate and an antimouse IgG-AuN conjugate sandwich a target mouse IgG with low nonspecific binding. Thus formed immunosensing complex is strongly attracted to an indium tin oxide (ITO) electrode modified with partially ferrocenyl-tethered dendrimers (Fc-Ds) by using an external magnet. The AuN of the immunosensing complex produces p-aminophenol from p-nitrophenol by catalytic reduction in the presence of NaBH(4), and the generated p-aminophenol is electrooxidized at the Fc-D-modified ITO electrode. The oxidized product, p-quinone imine, is reduced back to p-aminophenol by NaBH(4) and then re-electrooxidized at the electrode. This redox cycling greatly amplifies the electrochemical signal. Moreover, the Fc-D-modified ITO electrode exhibits a low background current. Accordingly, the high signal-to-background ratio allows an extremely low detection limit of 1 fg/mL (7 aM) in cyclic voltammetric experiments and, importantly, 100 ag/mL (0.7 aM) in differential pulse voltammetric experiments.     

Enhanced electron-transfer reactivity of horseradish peroxidase in phosphatidylcholine films and its catalysis to nitric oxide

Direct electrochemistry of horseradish peroxidase (HRP) embedded in film of phosphatidylcholine (PC) is investigated at a pyrolytic graphite electrode by voltammetric methods. The electron-transfer reactivity between incorporated HRP and the electrode is found to be greatly enhanced by phosphatidylcholine film. Cyclic voltammetry (CV) of this incorporated peroxidase shows a pair of well-defined and nearly reversible peaks, and the cathodic and anodic peak potentials are located at about -0.261 and -0.180 V, respectively versus saturated calomel electrode at pH 5.5. Ultraviolet-visible absorption spectra indicate that the heme microenvironment of HRP in phosphatidylcholine film is similar to that of its native status. It is also observed that HRP modified electrode is able to catalyze the electrochemical reduction of nitric oxide. Experimental results reveal that the peak current related to nitric oxide reduction is linearly proportional to its concentration in the ranges of 2.0 x 10(-7) -5.0 x 10(-6) mol (-1) and 2.0 x 10(-5) -1.0 x 10(-4) mol(-1), based on which an unmediated biosensor for nitric oxide is developed.     

Melittin as model system for probing interactions between proteins and cyclodextrins

Cylcodextrin sugars are cyclic sugars that have a hydrophilic exterior and a hydrophobic center. This enables cyclodextrins to solubilize hydrophobic molecules in aqueous media. Cyclodextrins may inhibit aggregation by intercalating surface aromatic residues and competing with interprotein aromatic clusters (pi-pi interactions). In order to investigate this concept, the interaction of hydroxypropyl-beta-cyclodextrin (HPBCD) with melittin is studied with steady-state and time-resolved fluorescence, fluorescence polarization, circular dichroism, and IR spectroscopy. HPBCD inhibits the aggregation of melittin. This inhibition and the spectroscopic results are consistent with the lone aromatic tryptophan of the peptide being intercalated within HPBCD.     

Electrochemical Properties As A Function of Ph for the Benzotriazine DI-N-Oxides

The electrochemistry of five benzotriazine di-N-oxides has been examined by cyclic voltammetry and differential pulse and dc polarographies as a function of pH. Between the pH range 8.5 and 2 the trend to less negative potentials with lowering of pH can be described by an equation of the type Ep = - apH + b. Comparison has been made with the mono- and zero-N-oxides which were found to show virtually identical trends in electron affinity with pH. The general electrochemical characteristics for the di- and mono-N-oxides under acidic conditions were found to be comparable with the zero-N-oxide. This was particularly the case on repeat scanning in the cyclic voltammetric mode. The redox mechanism involved reduction by a 4-electron addition step and subsequent loss of the A-oxide group(s) yielding the intact benzotriazine heterocycle. The helerocycle was also redox active, involving a reversible 2-electron reduction. for the di-N-oxides these two stages could be identified as separate processes at alkaline pH, but ony a single step at acidic values. The mono-N-oxide in which the electrochemical behaviour was dominated by the triazine, showed only a single reduction step, although the single N-oxide group was redox active.     

Characterization and electrocatalytic properties of Prussian blue electrochemically deposited on nano-Au/PAMAM dendrimer-modified gold electrode

Gold electrode was modified with 3-mercaptopropionic acid (MPA) and further reacted with poly(amidoamine) (PAMAM) dendrimer (generation 4.0) then attached the nano-Au to obtain films on which Prussian blue (PB) was electrochemically deposited to afford much wider pH adaptive range, much better electrochemical stability and excellent electrochemical response. The microstructure and electrochemical behavior of Au/MPA/PAMAM/nano-Au/PB electrode were investigated by scanning electron microscopy (SEM) and cyclic voltammetry. The electrochemical response of the Au/MPA/PAMAM/nano-Au/PB-modified electrode for the electrocatalytic reduction of hydrogen peroxide was investigated, and it was found that the sensitivity as well as the corresponding detection limits were improved as compared to the voltammetric response of a Au/PB-modified electrode and Au/MPA/PAMAM/PB electrode. Based on this, a new electrochemical sensor for determination of hydrogen peroxide has been developed.     

Caffeine, pentoxifylline and theophylline form stacking complexes with IQ-type heterocyclic aromatic amines

Methylxanthines (MTX), in particular caffeine (CAF), are known as the most widely consumed alkaloids worldwide. Many accumulated statistical data indicate the protective effect of CAF intake against several types of cancer. One of the possible explanations of this phenomenon is direct non-covalent interaction between CAF and aromatic mutagen/carcinogen molecules through stacking (π-π) complexes formation. Here we demonstrate that CAF and other MTX, pentoxifylline (PTX) and theophylline (TH), form stacking complexes with carcinogenic imidazoquinoline-type (IQ-type) food-borne heterocyclic aromatic amines (HCAs). We estimated neighborhood association constants (K_AC of the order of magnitude of 10² M⁻¹) in neutral and acidic environment and enthalpy changes (ΔH values between −15.1 and −39.8 kJ/mol) for these interactions using UV-Vis spectroscopy, calculations based on thermodynamical model of mixed aggregation and titration microcalorimetry. Moreover, using Ames test with Salmonella typhimurium TA98 strain and recently developed mutagenicity assay based on bioluminescence of Vibrio harveyi A16 strain, we demonstrated a statistically significant reduction in HCAs mutagenic activity in the presence of MTX.