Electrochemical detection and discrimination of single copy gene target DNA in non-amplified genomic DNA

A sensitive, fast and inexpensive method for direct electrochemical detection of target DNA sequences in non-amplified genomic DNA samples is described. Hybridization detection relies on the alteration in guanine oxidation signal following hybridization of the probe with complementary genomic DNA. Initially, the method was tested to detect target DNA on low cycle number PCR amplicons. Having obtained promising detection results from only 5 cycles product, the feasibility of target sequence detection in extracted genomic DNA without PCR amplification, but with the vortex mediated fragmentation of the large genomic DNA into small pieces was examined. Experimental variables affecting the efficiency of sensor were investigated. Detection experiments with various non-complementary genomic DNAs as well as a proper probe, non-specific with respect to all genomic samples confirmed the excellent selectivity of the approach. The sensitivity of the method for analyzing the vortex mediated fragmentized genomic DNA samples is estimated to be approximately of 0.58 ng/μl.     

Ru(bpy)3-doped silica nanoparticle DNA probe for the electrogenerated chemiluminescence detection of DNA hybridization

A sensitive electrogenerated chemiluminescence (ECL) detection of DNA hybridization, based on tris(2,2′-bipyridyl)ruthenium(II)-doped silica nanoparticles (Ru(bpy)₃²⁺-doped SNPs) as DNA tags, is described. In this protocol, Ru(bpy)₃²⁺-doped SNPs was used for DNA labeling with trimethoxysilylpropydiethylenetriamine(DETA) and glutaraldehyde as linking agents. The Ru(bpy)₃²⁺-doped SNPs labeled DNA probe was hybridized with target DNA immobilized on the surface of polypyrrole (PPy) modified Pt electrode. The hybridization events were evaluated by ECL measurements and only the complementary sequence could form a double-stranded DNA (dsDNA) with DNA probe and give strong ECL signals. A three-base mismatch sequence and a non-complementary sequence had almost negligible responses. Due to the large number of Ru(bpy)₃²⁺ molecules inside SNPs, the assay allows detection at levels as low as 1.0 × 10⁻¹³ mol l⁻¹ of the target DNA. The intensity of ECL was linearly related to the concentration of the complementary sequence in the range of 2.0 × 10⁻¹³ to 2.0 × 10⁻⁹ mol l⁻¹.     

Improvement of the growth of Arthrospira (Spirulina) platensis from Toliara (Madagascar): Effect of agitation, salinity and CO2 addition

Arthrospira (Spirulina) platensis Toliara isolated from alkaline and salt lakes in the south-western area of Madagascar is a potential source of proteins that could efficiently fight against food deficiency in developing countries like Madagascar. Up to now, productivity in this country has been low, so a better understanding of the growth conditions of this species is needed to improve its production. Growth experiments were undertaken in bubble columns at laboratory scale. The influence of agitation of the culture, medium salinity (ranging from 13 to 35 g L⁻¹) and CO₂ addition (ranging from 0 to 2%, v/v) on growth and protein content was examined. Because Arthrospira cells are fragile, a bubble column without additional mixing gave the best growth. Arthrospira (Spirulina) platensis showed higher specific growth rate (μ_max) and protein content for lower salinity. Addition of 1% of CO₂ improved the productivity by near 60%. The feasability of semi-continuous culture was demonstrated and optimal culture conditions led to a mean productivity of 0.22 ± 0.03 g L⁻¹ d⁻¹, a mean specific growth rate of 0.015 ± 0.002 h⁻¹ and a protein content of 53 ± 2% of total dry weight.     

DNA hybridization biosensor using chitosan–carbon nanotubes composite film as an immobilization platform and [Cu(bpy)(MBZ)2(H2O)] (bpy = 2,2′-bipyridine, MBZ = p-methylbenzoate) as a novel redox indicator

In this paper, a new DNA hybridization detection strategy was developed based on the immobilization of capture probe DNA on a chitosan (CS)–carbon nanotubes (CNTs) composite modified glassy carbon electrode (CS–CNTs/GCE) and the use of a copper complex, [Cu(bpy)(MBZ)₂(H₂O)] (bpy = 2,2′-bipyridine, MBZ = p-methylbenzoate), as a new redox hybridization indicator. The electrochemical characterization experiments showed that the nanocomposite film of CS–CNTs could effectively immobilize the capture probe DNA and greatly improve the electron-transfer reactions of the electroactive molecules. Electrochemical and fluorescent spectroscopic analysis revealed that the polypyridyl copper complex of [Cu(bpy)(MBZ)₂(H₂O)] bound to DNA via a typical intercalation mode. Surface studies further showed that the copper complex can discriminate between double-stranded and single-stranded DNA that immobilized on the surface of CS–CNTs/GCE. When being utilized as a redox indicator for the detection of hybridization for short DNA species related to phosphinothricin acetyltransferase (PAT), the indicator showed good specificity for recognizing the complementary, three-base mismatched and non-complementary DNA. Under the optimized conditions, the oxidation peak currents of the copper complex enhanced linearly with increases in the concentration of the complementary sequence in the range from 5.0 × 10⁻¹⁰ to 1.0 × 10⁻⁸ M. A detection limit of 5.0 × 10⁻¹⁰ M was also obtained based on the constructed DNA biosensor.     

Synthesis of Ethyl 5 Z ,9 Z ,12 Z -octadecatrienoate (ethyl pinolenate) and Methyl 12 Z ,15 Z -octadecadienoate

Pinolenic acid (5Z,9Z,12Z-octadecatrienoic acid, 1a), one of the most abundant trienoic fatty acids in nature, is very difficult to obtain in quantity in a pure state from the highly complex mixture of unsaturated tall oil fatty acids. For this reason its chemistry has been little studied when compared to linolenic or linoleic acids. A simple synthesis of esters of 1a and of 12Z,15Z-octadecadienoic acid 3 using the one pot double Wittig procedure is described here. The products of double Wittig reactions were purified by argentation chromatography, and their structural purity was established by ¹H-, ¹³C-NMR and 2D-NMR spectroscopies.

Electrocatalytic tandem Knoevenagel–Michael addition of barbituric acids to isatins: Facile and efficient way to substituted 5,5′-(2-oxo-2,3-dihydro-1H-indole-3,3-diyl)bis(pyrimidine-2,4,6-(1H,3H,5H)-trione) scaffold

Electrocatalytic transformation of isatins and barbituric acids in ethanol in an undivided cell in the presence of sodium bromide as an electrolyte results in the formation of substituted 5,5′-(2-oxo-2,3-dihydro-1H-indole-3,3-diyl)bis(pyrimidine-2,4,6(1H,3H,5H)-triones) in 89–95% substance yields and 890–950% current yields. The developed efficient electrocatalytic approach to medicinally relevant 5,5′-(2-oxo-2,3-dihydro-1H-indole-3,3-diyl)bis(pyrimidine-2,4,6(1H,3H,5H)-trione) scaffold is 10 times faster than general chemical method, is beneficial from the viewpoint of diversity-oriented large-scale processes and represents novel example of facile environmentally benign synthetic concept for electrocatalytic tandem reaction strategy.     

An experimental and computational study of intramolecular charge transfer: Diarylamino derivatives of 7H-benzimidazo(2,1-a)benz(d,e)isoquinolin-7-ones

A novel intramolecular donor–acceptor system of four isomers consisting of 7H-benzimidazo(2,1-a)benz(d,e)isoquinolin-7-ones and diarylamine units was synthesized and characterized; the absorption and fluorescence spectra of the system in a variety of solvents were investigated. Intramolecular charge transfer was confirmed within the system by virtue of shifts in emission maximum with increasing solvent polarity; a high dipole moment for the intramolecular excited state was calculated using the Lippert equation. Shorter lifetimes were observed in polar solvents compared with those in non-polar solvents, indicating strong dipole–dipole interactions occurred. The ground-state geometry, lowest energy transition and the UV–vis spectrum of the system were studied using density functional theory and time-dependent density functional theory at B3LYP/6-31G^* level, which showed that the calculated outcomes were in good agreement with experimental data.     

Electrochemical biosensor based on the interaction between copper(II) complex with 4,5-diazafluorene-9-one and bromine ligands and deoxyribonucleic acid

The copper complex of 4,5-diazafluorene-9-one (dafone) and bromine ligands ([Cu(dafone)₂]Br₂) was prepared and its interaction with double-stranded salmon sperm DNA (dsDNA) in pH 8.0 Britton-Robinson (B-R) buffer solution was studied by electrochemical experiments at the glassy carbon electrode (GCE). It was revealed that Cu(dafone)₂Br₂ could bind with salmon sperm DNA strands mainly by intercalation mode. The binding number of [Cu(dafone)₂]Br₂ for each salmon sperm dsDNA chain and equilibrium constant of the binding reaction were calculated to be 3 and 2.8 × 10¹² L³ mol⁻³, respectively. The Cu(dafone)₂Br₂ was further utilized as a new electrochemical DNA indicator for the detection of human hepatitis B virus (HBV) DNA fragment by differential pulse voltammetry (DPV). The difference of its electrochemical responses occurred between hybridized dsDNA duplex and probe DNA was explored to assess the selectivity of the developed electrochemical DNA biosensor. The constructed electrochemical DNA biosensor achieved a detection limit of 3.18 × 10⁻⁹ mol L⁻¹ for complementary target DNA and also realized a robust stability and good reusability.     

Electrochemical preparation of self-doped poly(N-(3-sulfonicpropion) anilide) and its application in sensing ethanol

A novel containing sulfonic acid group aniline monomer, N-(3-sulfonicpropion) anilide, was synthesized in three steps and subsequently to be electropolymerized on a glassy carbon electrode (GCE). The resulting self-doped poly(N-(3-sulfonicpropion) anilide) (SPAN/GCE) held 79.5% electrochemical activity when transferred from 0.1 M pH 7.0 PBS to 0.1 M pH 10.0 PBS, indicating its remarkable extension of the redox activity. The SPAN/GCE was characterized by X-ray photoelectron spectroscopy (XPS) and found that 30% of the nitrogen atoms are sulfonated. Preliminary experimental results show that after the immobilization of alcohol dehydrogenase (ADH) on the SPAN/GCE (ADH/SPAN/GCE), the ADH/SPAN/GCE showed good electrocatalytic activity toward the oxidation of ethanol. These reveal that the SPAN/GCE is quite promising in the fields of biosensors, biofuel cells and other bioelectrochemical devices.     

Predicting toxicity of tall larkspur (Delphinium barbeyi): measurement of the variation in alkaloid concentration among plants and among years

Tall larkspur (Delphinium barbeyi) is the principal mountain larkspur responsible for the majority of cattle deaths on mountain rangelands in western Colorado and central and southern Utah in the United States. Ten plants in each of two tall larkspur populations in the mountains near Ferron and Salina, Utah, were marked, and single stalks were harvested periodically through the growing season for 4 yr. Toxic alkaloid concentration [alkaloids containing the N-(methylsuccimimido)-anthranilik ester group] was determined by Fourier transform infrared (FTIR) spectroscopy. Individual larkspur plants varied in alkaloid concentrations, especially in early growth (14–38 mg/g). As the concentration declined over the growing season, variation among plants also declined. There were yearly differences in alkaloid concentration among individual plants (P < 0.01) and populations (P < 0.001), even after accounting for differences in phenological growth between years. Variables such as precipitation, temperature, days since snow melt, growing degree days (sum of mean temperature each day from snow melt), and plant height and weight were all considered in a Mallows Cp multiple regression selection procedure to predict alkaloid concentration. The mixed model procedure in SAS adjusted the regression equation for locations and years. Growing degree days was the best single predictor of alkaloid levels: ln y = (3.581 – 0.00423 GDD), R ² = 0.85. Internal validation of this equation within individual years and locations from which the equation was developed, produced correlations between observed versus predicted values ranging from r = 0.73 to 0.93. External validations on nine other larkspur populations produced correlations ranging from r = 0.76 to 0.99. This predictive equation can provide a tool for ranchers and land managers to make management decisions of when to graze cattle in larkspur areas.     

N-(triphenylphosphoranylidene) aniline as a novel electrolyte additive for high voltage LiCoO2 operations in lithium ion batteries

We report N-(triphenylphosphoranylidene) aniline (TPPA) as a new electrolyte additive for high performance lithium cobalt oxide (LiCoO₂) electrodes during high voltage operations. When cycled in the voltage range of 3.0–4.4 V, graphite-LiCoO₂ full cells with 0.2 wt% TPPA exhibited 10% increased discharge capacities after 200 cycles compared to those of control cells with no such additive. The enhanced cycling performance is attributed to the additive effect toward the modified surface films on LiCoO₂ electrodes that suppress the decomposition of both solvent and salt in the electrolyte. This additive effect was characterized by electrochemical impedance spectroscopy (EIS) and X-ray photoelectron spectroscopy (XPS).     

Scanning electrochemical microscopy studies of micropatterned copper sulfide (CuxS) thin films fabricated by a wet chemistry method

Patterned copper sulfide (Cu_xS) microstructures on Si (1 1 1) wafers were successfully fabricated by a relatively simple solution growth method using copper sulfate, ethylenediaminetetraacetate and sodium thiosulfate aqueous solutions as precursors. The Cu_xS particles were selectively deposited on a patterned self-assembled monolayer of 3-aminopropyltriethoxysilane regions created by photolithography. To obtain high quality Cu_xS films, preparative conditions such as concentration, proportion, pH and temperature of the precursor solutions were optimized. Various techniques such as optical microscopy, atomic force microscopy (AFM), X-ray diffraction, optical absorption and scanning electrochemical microscopy (SECM) were employed to examine the topography and properties of the micro-patterned Cu_xS films. Optical microscopy and AFM results indicated that the Cu_xS micro-pattern possessed high selectivity and clear edge resolution. From combined X-ray diffraction analysis and optical band gap calculations we conclude that Cu₉S₅ (digenite) was the main phase within the resultant Cu_xS film. Both SECM image and cyclic voltammograms confirmed that the Cu_xS film had good electrical conductivity. Moreover, from SECM approach curve analysis, the apparent electron-transfer rate constant (k) in the micro-pattern of Cu_xS dominated surface was estimated as 0.04 cm/s. The SECM current map showed high edge acuity of the micro-patterned Cu_xS.     

A DNA electrochemical sensor with poly-l-lysine/single-walled carbon nanotubes films and its application for the highly sensitive EIS detection of PAT gene fragment and PCR amplification of NOS gene

A sensitive and novel DNA electrochemical biosensor for the detection of the transgenic plants gene fragment by electrochemical impedance spectroscopy (EIS) was presented. The well-dispersed carboxylic group-functionalized single-walled carbon nanotubes (SWNTs) were dripped onto the carbon paste electrode (CPE) surface firstly, and poly-l-lysine films (pLys) were subsequently electropolymerized by cyclic voltammetry (CV) to prepare pLys/SWNTs/CPE. The morphology of pLys/SWNTs films was examined using a field emission scanning electron microscope (SEM). The pLys/SWNTs films modified electrode exhibited very good conductivity. DNA probes were easily immobilized on the poly-l-lysine films via electrostatic adsorption. The hybridization events were monitored with electrochemical impedance spectroscopy using [Fe(CN)₆]^3−/4− as indicator. The PAT gene fragment from phosphinothricin acetyltransferase gene was detected by this DNA electrochemical sensor. The dynamic detection range of this sensor to the PAT gene fragment was from 1.0 × 10⁻¹² to 1.0 × 10⁻⁷ mol/L. A detection limit of 3.1 × 10⁻¹³ mol/L could be estimated. The PCR amplification of NOS gene from the sample of a kind of transgenic modified bean was also detected satisfactorily by EIS.     

Process research of macroporous resin chromotography for separation of N-(p-coumaroyl)serotonin and N-feruloylserotonin from Chinese safflower seed extracts

N-(p-Coumaroyl)serotonin (CS) and N-feruloylserotonin (FS) are two bioactive serotonin derivatives in safflower (Carthamus tinctorius L.) seeds with many biological effects. In the present study, the sorption and desorption characteristics of six widely used macroporous resins, coded D312, 860021, DM131, HZ801, AB-8 and XDA-1, respectively, is critically evaluated and compared for enrichment and preparative separation of CS and FS. Static adsorption and desorption experiments on these resins showed that XDA-1 had the best adsorption and desorption equilibrium for CS and FS, and its adsorption equilibrium fits the best to the Langmuir isotherm. Dynamic adsorption and desorption experiments on XDA-1 resin packed column were conducted to establish the optimum parameters as: CS and FS concentration in sample solution 0.346 and 0.276 mg/mL, respectively, sample size 5-bed volumes (BV), sample flow rate 2 mL/min, temperature 25 °C (for adsorption); eluent 80% ethanol, 11 BV, flow rate 1 mL/min (for desorption). After one run of adsorption and desorption, the contents of CS and FS were increased from 2.01%, 1.67% to 24.1% and 22.4%, and the recoveries were 77.2% and 68.5%, respectively. The chromatographic process optimized in the present work is a promising basis for large scale preparation of CS and FS upon further scaling up tests.     

Electrochemical detection of DNA hybridization using a water-soluble branched polyethyleneimine-cobalt(III)-phenanthroline indicator and PNA probe on Au electrodes

An electrochemical method for the detection of DNA hybridization using a novel electroactive, cationic, and water-soluble branched polyethyleneimine (BPEI)-cobalt(III)-phenanthroline(phen) polymeric indicator and single-stranded neutral peptide nucleic acid (PNA) probe on the Au electrode was developed. The indicator possesses some free amine groups, as well as cationic cobalt complexes in the polymer chain. It does not bind to neutral PNA capture probe alone. However, the indicator strongly interacts with the negatively charged backbone of the complementary oligonucleotide bound to the PNA probe through electrostatic interactions. The coordination spherical moieties also interact with the probe by embedding into the double-helix structure of PNA-DNA. These two interactions enable transduction of hybridization, producing a clear electrical signal in differential pulse voltammetry (DPV). The correlation against non-complementary DNA, three-base and one-base mismatch DNA was sharp, and the signal of indicator for the target DNA demonstrated a linear relationship within the concentration range of 5.0 × 10⁻⁹ to 2.5 × 10⁻⁷ M (R = 0.9940) with a detection limit of 5.6 × 10⁻¹⁰ M. These studies showed that the novel polymeric indicator and single-stranded PNA probe could be used to fabricate an electrochemical biosensor for DNA detection. This technique can provide an alternative route for expanding the range of detection methods available for DNA hybridization.     

Copolymers of pyrrole and N-(hydroxypropyl)pyrrole: properties and interaction with DNA

Copolymers derived from mixtures of pyrrole and N-(hydroxypropyl)pyrrole have been prepared electrochemically using various concentration ratios. Copolymers were generated on stainless electrodes by chronoamperometry and cyclic voltammetry in a LiClO₄ acetonitrile solution. Relevant physical (density and doping level) and electrochemical (electroactivity and electrostability) properties of the copolymers have been examined and compared with those of the two corresponding homopolymers, which were prepared using the same electrochemical procedures. Results show that the copolymer obtained using the 25:75 N-(hydroxypropyl)pyrrole:pyrrole molar ratio presents an interesting behavior. Finally, the ability of the latter copolymer to form specific interactions with plasmid DNA has been compared with that of polypyrrole.     

Synthesis, structure and magnetic properties of the first copper(II) complex with an (E)-4-aryl-4-oxo-2-butenoato ligand

A new binuclear Cu(II) complex with an (E)-4-(2,4-diisopropylphenyl)-4-oxo-2-butenoato ligand (L) was successfully synthesized and characterized by elemental analysis and IR-spectroscopy. The structures of (E)-4-(2,4-diisopropylphenyl)-4-oxo-2-butenoic acid (HL), and the corresponding (tetrakis)-μ-[(E)-4-(2,4-diisopropylphenyl)-4-oxo-2-butenoato]-bis(ethanol)-copper(II) complex, [Cu₂L₄(C₂H₅OH)₂], were determined by single crystal X-ray analyses and are preliminarily discussed. This is the first complex of a transition metal with ligand L, as well as the first determined crystal structure of a metal complex with this type of ligand. Analysis of the magnetic susceptibility measurements of the isolated [Cu₂L₄(C₂H₅OH)₂] · H₂O complex shows the existence of a strong anti-ferromagnetic intradimer coupling, with an exchange integral value 2J of −260 cm⁻¹.     

Electrochemical synthesis and characterization of poly(pyrrole-co-o-toluidine)

The electrochemical polymerization of o-toluidine has been investigated in oxalic acid solution. It was shown that the oxidation of monomer could be achieved but this process does not yield a stable, homogenous polymer film on either platinum or mild steel electrodes. Therefore the copolymerization between pyrrole and o-toluidine has been studied as an alternative method for obtaining good quality coating (low permeability and water mobility, high stability), which could also be easily synthesized on steel. For this aim, various monomer feed ratio solutions of pyrrole:o-toluidine 9:1, 8:2 and 7:3 have been examined, in aqueous oxalic acid solution. By using cyclic voltammetry technique, copolymer films were realized on platinum and steel, successfully. The temperature of synthesis solution was found to have a vital role on polymerization and film growth, as much as the monomer feed ratio. The synthesis of homogenous copolymer film could only be achieved under ≤25 °C conditions with using the 9:1 ratio, while the 8:2 ratios could only produce stable films below 5 °C. As the amount of o-toludine increased the required temperature value decreased further, 7:3 ratio could only give a stable copolymer film below 2 °C. The characterization of deposited copolymer coating has been realized by using SEM micrographs, UV–vis and FT-IR spectroscopy techniques and cyclic voltammetry. The protective behaviour of these coatings was also investigated against mild steel corrosion in 3.5% NaCl solution, by means of electrochemical impedance spectroscopy (EIS) and anodic polarization curves. It was found that the monomer feed 8:2 ratio gave the most effective coating against the corrosion of mild steel.     

Electrochemical insight into the mechanism of electron transport in biofilms of Geobacter sulfurreducens

Electroactive bacterial biofilms can be produced on a polarized electrode by forcing its use as the final electron acceptor for bacterial respiration. This strategy offers the researcher the unique possibility to control the respiration process with extreme precision. The production of current, the accumulation of charge and the conducting properties of electroactive biofilms has been interrogated in this work through very basic electrochemical techniques including chronopotentiometry, chronoamperometry and cyclic voltammetry. Presented results indicate that charge can be accumulated in the biofilm conductive network, that network conductivity does not represent a limit for current production and that both the steady state current and the amount of accumulated charge depend on the redox state of cytochromes wiring the cells to the electrode. A model of biofilm conduction is presented as well.     

Electrochemical properties of palladium(II) trans- and cis-diglycinate complexes

The kinetics of trans–cis-isomerization of palladium(II) diglycinate complexes have been studied by the spectrophotometric and potentiometric methods. The reaction of transition of trans-[Pd(Gly)₂] to cis-[Pd(Gly)₂] is described by a first-order equation. The half-life of the trans-isomer [Pd(Gly)₂] and the rate constant of the reaction of its transition to cis-isomeric structure have been determined. A mechanism of transformation of trans-[Pd(Gly)₂] into cis-[Pd(Gly)₂] is proposed. The spectral characteristics and electrochemical parameters of palladium(II) trans- and cis-diglycinate complexes have been determined. The mechanism of electroreduction of palladium(II) from glycinate electrolyte containing no excess free ligand has been elucidated.