Interaction of E6 Gene from Human Papilloma Virus 16 (HPV-16) with CdS Quantum Dots

The aim of this study was to analyze the interactions of blue and yellow fluorescent CdS quantum dots (CdS-QDs) with human papillomavirus 16 (HPV-16) oncogene E6. The interactions were investigated using chip capillary electrophoresis, spectrophotometry and square wave voltammetry (SWV). Using chip capillary electrophoresis we proved that blue fluorescent CdS-QDs (0.5 mM) caused an increase of the migration time of the E6 HPV-16 DNA–CdS-QDs complex by 42 s compared to control DNA (E6 HPV-16). The same concentration of yellow fluorescent CdS-QDs caused an increase in the migration time of the DNA–CdS-QDs complex by 108 s compared to the control DNA (E6 HPV-16). The difference in the migration times between both complexes was 66 s. Using square wave voltammetry (SWV), the reduction signal of cytosine and adenine (peak CA) was observed, after the complex with 2.5 µg mL^?1 DNA was formed. A decrease of the peak CA reduction signal of the complex DNA–CdS-QDs by 90 % was caused when yellow fluorescent CdS-QDs (0.03 mM) were used. The same concentration of blue fluorescent CdS-QDs caused only a 50 % decrease of the C and A reduction signal of the DNA–CdS-QDs complex. The difference between both CdS-QDs was 40 %. Electrochemical measurements and chip electrophoresis analyses confirmed that the yellow fluorescent CdS-QDs show higher affinity to the DNA (E6 HPV-16) compared to blue ones.     

Static and Hydrodynamic Monitoring of Citalopram Based on its Electro-oxidation Behavior at a Glassy-Carbon Surface

Abstract

The electrooxidative behavior of citalopram (CTL) in aqueous media was studied by cyclic voltammetry (CV) and square-wave voltammetry (SWV) at a glassy-carbon electrode. The electrochemical behaviour of CTL involves two electrons and two protons in the irreversible and diffusion controlled oxidation of the tertiary amine group. The maximum analytical signal was obtained in a phosphate buffer (pH = 8.2). For analytical purposes, an SWV method and a flow-injection analysis (FIA) system with amperometric detection were developed. The optimised SWV method showed a linear range between 1.10 × 10^?5–1.20 × 10^?4 mol L^?1, with a limit of detection (LOD) of 9.5 × 10^?6 mol L^?1. Using the FIA method, a linear range between 2.00 × 10^?6–9.00 × 10^?5 mol L^?1 and an LOD of 1.9 × 10^?6 mol L^?1 were obtained. The validation of both methods revealed good performance characteristics confirming applicability for the quantification of CTL in several pharmaceutical products.     

Electrochemical biosensor for sensitively simultaneous determination of dopamine, uric acid, guanine, and adenine based on poly-melamine and nano Ag hybridized film-modified electrode

An electrochemical sensor for simultaneous determination of dopamine (DA), uric acid (UA), guanine (G), and adenine (A) has been constructed by copolymerizing melamine monomer and Ag ions on a glassy carbon electrode (GCE) with cyclic voltammetry. The poly-melamine and nano Ag formed a hybridized film on the surface of the GCE. The morphology of the film was characterized by scanning electron microscope. The electrochemical and electrocatalytic properties of this film were characterized by cyclic voltammetry, linear sweep voltammetry, and square wave voltammetry (SWV). In 0.1 M phosphate buffer solution (pH 4.5), the modified electrode resolved the electrochemical response of DA, UA, G, and A into four well-defined voltammetric oxidation peaks by SWV; the oxidation peak current of DA, UA, G, and A increased 13-, 6-, 7-, and 9-fold, respectively, compared with those at the bare GCE and the SWV peak currents of DA, UA, G, and A with linear concentrations in the ranges of 0.1–50, 0.1–50, 0.1–50, and 0.1–60 μM, respectively. Based on this, a method for simultaneous determination of these species in mixture was setup. The detection limits were 10 nM for DA, 100 nM for UA, 8 nM for G, and 8 nM for A.     

DNA-silver Nanocluster Binary Probes for Ratiometric Fluorescent Detection of HPV-related DNA

Herein, we described a ratiometric strategy based on "chameleon" DNA-silver nanoclusters( DNA-AgNCs) fluorescent binary probes. The strategy was applied to detect high-risk human papillomavirus( HPV) DNA sequences, HPV-16. First, DNA-AgNCs were synthesized by a simple reduction method. The obtained nanoprobes showed typical yellow and red fluorescence of AgNCs. Upon the addition of HPV-16 DNA, the yellow fluorescence of AgNCs was reduced greatly, whereas tlie red fluorescence of AgNCs was increased. The concentration of HPV-16 DNA in the samples was characterized by the ratio of fluorescence intensity at 570 and 630 nm. Tlie ratiometric nanoprobes showed good selectivity for HPV-16 DNA, and the detection limit was 2 ninol/L. In addition, the practical applicability of this strategy was demonstrated by analysing the HPV-16 DNA in hiunan serum, illustrating its potential promise for clinical diagnosis.     

Quantitative Analysis of Irbesartan in Pharmaceuticals and Human Biological Fluids by Voltammetry

Abstract

A differential pulse (DP) and square wave (SW) voltammetric techniques were developed for the determination of irbesartan. The electrochemical behavior of irbesartan was investigated by cyclic voltammetry (CV), differential pulse voltammetry (DPV), and square wave voltammetry (SWV) at the hanging mercury drop electrode (HMDE). Different parameters were tested to optimize the conditions of the determination. It was found that in the range of 8 × 10^?6–1 × 10^?4 M, the currents measured by both of methods presented a good linear property as a function of the concentration of irbesartan. In addition, validation parameters, such as reproducibility, sensitivity, and recovery were evaluated as well. The slope of the log Ip- log ν linear plot was 0.58 indicating the diffusion control for 0.5 M sulphuric acid without the need for separation or complex sample preparation, since there was no interference from the excipients and endogenous substances.

The methods were successfully applied to the analysis of irbesartan in the pharmaceutical tablet formulations and in human serum samples.     

Comparison of Boron-Doped Diamond and Glassy Carbon Electrodes for Determination of Terbinafine in Pharmaceuticals Using Differential Pulse and Square Wave Voltammetry

This paper examines the electrochemical oxidation of terbinafine with the boron doped diamond and glassy carbon electrodes. The studies were performed by cyclic voltammetry (CV), differential pulse voltammetry (DPV), and square-wave voltammetry (SWV). The supporting electrolytes, solution pH, the range of potentials, and the scan rates were optimized. Terbinafine was irreversibly oxidized in all electrolytes, yielding well-defined peaks in the positive potential range. The peak potential shifted towards less positive values as the solution pH increased. Voltammetric determination of terbinafine was performed under the optimized conditions. Using the boron doped diamond electrode, a linear relationship between current and concentration was obtained between 5.44 × 10^?7 and 5.18 and 10^?6 mol/L with SWV and between 7.75 · 10^?7 and 8.55 · 10^?6 mol/L by DPV. At the glassy carbon electrode, a linear relationship between 7.75 · 10^?7 and 8.55 · 10^?6 mol/L was obtained by SWV and between 7.75 · 10^?7 and 1.05 · 10^?5 mol/L by DPV. The sensitivity, precision, and selectivity of the procedures were evaluated. In order to check the practical application of the developed methods, the concentration of terbinafine was determined in pharmaceutical preparations.     

Response surface methodology for optimisation of 4,6-Dinitro-o-cresol quantification using hanging mercury drop electrode

A method for the pesticide DNOC (4,6-Dinitro-o-cresol) quantification using hanging mercury drop electrode (HMDE) and stripping square wave voltammetry (SSWV) optimisation is proposed. As a continuous cathodic current decrease was observed during the experiments, a waiting time, together with solution pH and SWV instrumental variables were optimised by factorial designs. From the two reduction current peaks values, only one was considered as dependent variable in the optimisation process. While the cathodic current peak and standard deviation were used in the waiting time and solution pH optimisation process, the instrumental parameters for SWV were optimised by using only current peak values. With the optimal parameters, a calibration curve from (0.01–0.55) × 10^?6 mol L^?1 with LOD of 2 × 10^?8 mol L^?1 was obtained. The proposed method was checked for DNOC quantification in different water samples obtained from Cordoba area and very good results, with recovery values around 102% were observed.     

Mercury meniscus on solid silver amalgam electrode as a sensitive electrochemical sensor for tetrachlorvinphos

The in-house prepared mercury meniscus modified solid silver amalgam electrode (m-AgSAE) was successfully applied for the detection of organophosphate pesticide tetrachlorvinphos in pH 7 buffer solution. The electrochemical performance of m-AgSAE for the reduction of tetrachlorvinphos was evaluated using cyclic voltammetry (CV), differential pulse voltammetry (DPV), and square wave voltammetry (SWV), respectively. The surface morphology of solid silver electrode (AgE), as-amalgamated solid silver amalgam electrode (AgSAE), and polished solid silver amalgam electrode (p-AgSAE) was examined by field emission scanning electron microscopy (FESEM). Among the applied techniques, DPV and SWV analysis showed a remarkable increase in the reduction peak current and provided a simple, fast, and sensitive method for the determination of tetrachlorvinphos. The electrochemical impedance spectroscopy (EIS) was used to correlate the electrocatalytic activity of AgSAE, p-AgSAE and m-AgSAE with their interfacial charge transport capabilities. Under the optimized experimental conditions, the DPV and SWV responses were linear over the 1–9 μM and 10–50 μM concentration ranges with a detection limit of 0.06 μM for DPV and 0.04 for SWV. The estimation of tetrachlorvinphos in the ground and waste water samples with the proposed method was in good agreement with that of the added amount. The proposed electrochemical method not only extends the application of non-toxic m-AgSAE, but also offers new possibilities for fast and sensitive analysis of tetrachlorvinphos and its structural analogs in environmental samples.     

Synthesis and DNA studies of a copper(II) complex of 5,6-dihydro-5,6-epoxy-1,10-phenanthroline

A new copper(II) complex, [Cu(epoxy)(phen)(NO₃)](NO₃)·H₂O (epoxy = 5,6-dihydro-5,6-epoxy-1,10-phenanthroline, phen = 1,10-phenanthroline), has been synthesized and characterized by elemental analysis, IR and UV–vis spectroscopy, and single crystal X-ray diffraction. The complex crystallized in a monoclinic system with space group P21/n, a = 13.3199(8) Å, b = 11.2917(7) Å, c = 16.0832(9) Å, α = 90°, β = 107.827(4)°, and γ = 90°. The complex cation possesses a distorted octahedral geometry, with Jahn–Teller distortion occurring in the CuN₄O₂ core. The binding interaction with calf thymus DNA (CT–DNA) was investigated by UV–vis and fluorescence spectroscopy, cyclic voltammetry, and viscometry. The results support a partially intercalative binding mode. Efficient cleavage of plasmid DNA by the complex was observed by gel electrophoresis.     

Paramagnetic Particles Isolation of Influenza Oligonucleotide Labelled with CdS QDs

In this study, we describe hybridization design probes consisting of paramagnetic particles and quantum dots (QDs) with targeted DNA, and their application for detection of avian influenza virus (H5N1). Optical properties of QDs were beneficial, but the main attention was paid to the electroactivity of metal part of QDs and ODNs themselves. Differential pulse voltammetry was used for detection of cadmium(II) ions and square wave voltammetry for detection of cytosine–adenine peak in ODN-SH-Cd complex. It clearly follows from the obtained results that the optimized conditions were temperature of hybridization 25 °C, time of hybridization 35 min, and concentration of ODN-SH-Cd complex 20 μg mL^?1. The detection limit (3 signal/noise) was estimated as 15 ng mL^?1 of ODN-SH-Cd.     

Carbon paste electrode modified with Fe<Subscript>3</Subscript>O<Subscript>4</Subscript> nanoparticles and BMI.PF<Subscript>6</Subscript> ionic liquid for determination of estrone by square-wave voltammetry

A carbon paste electrode (CPE) modified with Fe₃O₄ nanoparticles (Fe₃O₄ NP) and the ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate (IL BMI.PF₆) was employed for the electroanalytical determination of estrone (E1) by square-wave voltammetry (SWV). At the modified electrode, cyclic voltammograms of E1 in B–R buffer (pH 12.0) showed an adsorption-controlled irreversible oxidation peak at around +0.365 V. The anodic current increased by a factor of five times and the peak potential shifted 65 mV to less positive values compared with the unmodified CPE. Under optimized conditions, the calibration curve obtained showed two linear ranges: from 4.0 to 9.0 μmol L^?1 and from 9.0 to 100.0 μmol L^?1. The limits of detection (LOD) and quantification (LOQ) attained were 0.47 and 4.0 μmol L^?1, respectively. The proposed modified electrode was applied to the determination of E1 in pork meat samples. Data provided by the proposed modified electrode were compared with data obtained by UV–vis spectroscopy. The outstanding performance of the electrochemical device indicates that Fe₃O₄ NP and the IL BMI.PF₆ are promising materials for the preparation of chemically modified electrodes for the determination of E1.     

Synthesis,structural characterization and DNA-binding studies of a new cobalt (II) complex: (HAcr)<Subscript>4</Subscript>[Co(Sal)<Subscript>3</Subscript>]

A new cobalt (II) coordination compound was synthesized using proton transfer mechanism. The reaction between CoCl₂·2H₂O, Salicylic acid (H₂Sal) and acridine (Acr) gave a new coordination compound formulated as (HAcr)₄[Co(Sal)₃], which was characterized by elemental analysis, NMR, IR and UV/Vis spectroscopies. The interaction of this complex with DNA has been investigated in vitro using UV absorption, fluorescence spectroscopy, viscosity measurements and gel electrophoresis methods. The intrinsic binding constant has been estimated to be 5.8 × 10⁵ M^?1 using UV absorption. The interaction of DNA–Co (II) complex caused quenching in fluorescence. The binding constant, the number of binding site and Stern–Volmer quenching constant have been calculated to be 7.7 × 10⁴ M^?1, 1.143 and 1.5 × 10⁴ Lmol^?1, respectively. The increase in the viscosity of DNA with increasing the concentration of the Co (II) complex and the observations of other experiments suggest that the cobalt (II) complex binds to DNA by partial intercalation binding mode. Furthermore, the interaction of DNA–Co (II) complex was confirmed using gel electrophoresis studies. Moreover, molecular docking technique predicted partial intercalation binding mode for the complex.     

An enzyme biosensor based on beta-galactosidase inhibition for electrochemical detection of cadmium (II) and chromium (VI)

The focus of this article is the development and optimisation of a simple, sensitive, selective biosensor for the detection of heavy metals, through inhibition of β-galactosidase (β-gal) from Aspergillus oryzae after its immobilisation on an electrochemical transducer by cross-linking with glutaraldehyde. The inhibition of β-gal by Cd(II) and Cr(VI) was demonstrated by the decrease of β-gal activity monitored by the conductometric signal. Under optimal conditions, the developed voltammetric and impedimetric biosensor, based on the specific interaction of metallic ions with beta-galactosidase that increases the electron transfer of the redox probe, presented a wide range of linearity. Detection limits of 3.12 × 10^–10 M (91.7 ng/L) were obtained, using both techniques, for Cr(VI). For Cd(II) detection limits were 2.85 × 10^–8 M (6.95 µg/L) using EIS and 3.22 × 10^–11 M (7.61 ng/L) using square wave voltammetry (SWV).     

Ultrasensitive Electrochemical Biosensor for HPV16 Oncogene Based on Y-shaped DNA Catalytic Hairpin Assembly and Template-free DNA Extension Reaction

An ultrasensitive electrochemical biosensor for HPV16 oncogene was explored. Hairpin DNA-1, which can specifically bind with HPV16 oncogene, was fixed on the surface of gold electrode. Two hairpin DNAs underwent catalytic hairpin assembling with hairpin DNA-1 to construct Y-shaped DNA nanostructure, liberating HPV16 oncogene for target recycling. The 3’ terminus of Y-shaped DNA nanostructure was prolonged under the catalysis of terminal deoxynucleotidyl transferase. Methylene blue was adsorbed onto DNA nanostructure to generate characteristic differential pulse voltammetry signal. This signal was increased with the concentration of HPV16 oncogene, and the detection limit of HPV16 oncogene was as low as 0.19 fM.     

A Sensitive Nanoporous Gold-Based Electrochemical DNA Biosensor for Escherichia coli Detection

A sensitive electrochemical DNA biosensor based on a mixed monolayer structure self-assembled at nanoporous gold (NPG) electrode surface was prepared for Escherichia coli (E. coli) detection. The NPG was fabricated on gold electrode, onto which thiolated oligonucleotides (SH-DNA) and mercaptohexanol (MCH) were covalently linked forming a mixed self-assembled monolayer (SAM). The hybridization between the SH-DNA/MCH modified biosensor and E. coli DNA was monitored with differential pulse voltammetry measurement using methylene blue (MB) as the hybridization indicator. The biosensor can detect 1 × 10^?12 M DNA target and 50 cfu/μL E. coli without any nucleic acid amplification steps. The detection limit was lowered to 50 cfu/mL after 5.0 h of incubation.     

Label-Free DNA Biosensor for Electrochemical Detection of Short DNA Sequences Related to Human Papilloma Virus

Abstract

Highly sensitive label-free techniques of DNA determination are particularly interesting in relation to the present development of an electrochemical hybridization biosensor for the detection of short DNA fragments specific to the human papilloma virus (HPV). Unlabeled DNA probes have been immobilized by spontaneous coadsorption of thiolated single-stranded oligonucleotides (HS-ssDNA) onto the sensing surface of a screen-printed gold electrode (SPGE). The covalently immobilized single-stranded DNA probe (HS-ssDNA) could selectively hybridize with its complementary DNA (cDNA) in solution to form double-stranded DNA (dsDNA) on the surface. DNA is treated with acid (e.g., 0.5 M chloridric acid), and the acid-released purine bases are directly determined by square wave voltammetry (SWV).

Variables of the probe-immobilization and hybridization steps are optimized to offer convenient quantitation of HPV DNA target, in connection with a short hybridization time. Peak currents were found to increase in the following order: hybrid-modified SPGE, 11-base mismatched modified SPGE, 18-base mismatched SPGE, and the probe modified SPGE. Control experiments with noncomplementary oligonucleotides were carried out to assess whether the suggested DNA sensor responds selectively to the target. The effect of the target DNA concentration on the hybridization signal was also studied. Under optimal conditions, this sensor has a good calibration range with HPV DNA sequence detection limit of 2 pg · ml^?1 (S/N = 3).     

Expression,Purification, and Characterization of a Recombinant Methionine Adenosyltransferase pDS16 in Pichia pastoris

Methionine adenosyltransferase (MAT, EC2.5.1.6) catalyzes the synthesis of S-adenosylmethionine (SAM) using l-methionine and adenosine triphosphate (ATP) as substrates. The mutant MAT pDS16 was obtained through DNA shuffling previously in our lab. Overexpression of pDS16 in Pichia pastoris led to about 65 % increase of MAT activity and SAM accumulation, compared with the strain overexpressing Saccharomyces cerevisiae MAT gene SAM2. Different strategies were tested to facilitate the expression and purification of pDS16. However, addition of the hexahistidine tag to pDS16 was shown to decrease the enzyme activity, and the yeast α-factor signal sequence could not effectivley direct the secretion of pDS16. The intracellular pDS16 was purified by a simple two-step procedure combining an ion exchange and hydrophobic interaction chromatography. Protein purity was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis to be 93 %, with the specific activity of 1.828 U/mg. Two-dimensional electrophoresis revealed pI of ～5.5. The purified enzyme followed Michaelis kinetics with a Km of 1.72 and 0.85 mM, and Vmax of 1.54 and 1.15 μmol/min/mg for ATP and L-methionine, respectively. pDS16 exhibited optimal activity at pH 8.5 and 45 °C with the requirement of divalent cation Mg²⁺ and was slightly stimulated by the monovalent cation K⁺. It showed an improved thermostability, about 50 % of the enzyme activity was retained even after preincubation at 50 °C for 2 h.     

Synthesis,characterization, and DNA binding of two copper(II) complexes as DNA fluorescent probes

[Cu(DAPT)₂Cl]Cl·H₂O and [Cu(DBM)(DAPT)Cl] [DAPT = 2,4-diamine-6-(pyrazin-2-yl)-1,3,5-triazine] were synthesized and characterized by IR and UV spectroscopy, elemental analysis, TG–DTA, molar conductivity, and LC–MS. The interaction with calf thymus DNA (ct-DNA) of the two complexes has been studied using UV spectra, fluorescent spectra, cyclic voltammetry, and viscosity measurements. The complexes interact with ct-DNA through classical intercalation. Fluorescence intensity changes of 1 and 2 in the absence and presence of ct-DNA have been investigated for quantitative determination of ct-DNA with the limit of detection of 3.8 and 7.7 ng mL^?1, respectively. From the result, the two complexes are potentially sensitive DNA fluorescent probes.     

Electrochemical Determination of Boron in Natural Waters

Abstract

A sensitive voltammetric method for the determination of trace boron, based on the formation of the complex of boric acid with 4‐hydroxy‐5‐[salicylideneamino]‐2‐7‐naphthalenedisulfonic acid (azomethine H) is described. The reduction of the boric acid‐azomethine H complex at a hanging mercury drop electrode was exploited by square wave voltammetry (SWV) and cyclic voltammetry to determine boron in natural water samples, which were collected in the regions surrounding the boron mines of Central Anatolia. A reduction peak that belongs to the boric acid‐azomethine H complex at this electrode was observed at ?1.05 V vs. Ag/AgCl/KCl_(sat.). The effects of various parameters, such as ligand concentration, boric acid concentration, and formation time of the boric acid‐azomethine H complex, were investigated. Electrochemical experiments were conducted in 1.0 M HOAc/0.5 M NH₄OAc buffer at pH of 4.4±0.2. Linear working range was established by regression analysis between 5.0×10^?8 M and 1.0×10^?4 M. The probable metal cation interferences in water samples were eliminated by adding EDTA (ethylenediaminetetraacetic acid) to the samples. Data obtained using the square wave voltammetric (SWV) technique was compared statistically with inductively coupled plasma mass spectroscopy (ICP‐MS) data. Evaluation of the method based on statistical data was performed and the values of the limit of detection (LOD) and limit of quantitation (LOQ) were found to be 4.17×10^?6 M and 1.39×10^?5 M, respectively.     

Sol–gel derived dye-bridged hybrid materials for white luminescence

Novel yellow and blue emissive dyes have been synthesized using 2,5-diamino-3,6-dicyanopyrazine and various alkoxysilanes and they are covalently bridged to cycloaliphatic epoxy functional oligosiloxane via non-hydrolytic sol–gel reaction. Dye-bridged hybrid materials (DBH) are fabricated by thermal curing the dye-bridged oligosiloxane. Structure and formation of dyes and siloxane network is studied using analysis method. Four components of red, yellow, green and blue emitting DBH cover entire visible range and white luminescence with high color rendering index is realized by controlling their combinations. We have ensured superior thermal stability DBH at 120 °C for 200 h caused by covalently bridged structure and robust siloxane matrix.