Double-stranded Calf Thymus DNA Research Articles

Impedimetric evaluation of hybrid cationic porphyrin/quantum dot multilayer assemblies: a biocompatible interface for calf thymus DNA immobilization

In this work, a cationic porphyrin, ascribed as [Ttolyl(P-(C6H5)3)4]4+, where Ttolyl = 5,10,15,20 tetrakistolylporphyrin, was electrostatically assembled with CdTe quantum dots capped with glutathione (GSH) of diameter ~3.28 nm via a layer-by-layer methodology. This multilayer assembly was evaluated as a biocompatible interface with synergic effects for calf thymus double-stranded DNA (CT DNA) immobilization, considering that the hybrid assembly contains a cationic porphyrin with a high binding constant for CT DNA, and quantum dots with polypeptide as a capping agent. The multilayer assembly, ascribed as ITO/{[Ttolyl(P-(C6H5)3)4]4+/CdTe} n (n = 1–5), was characterized by UV-Vis spectroscopy, cyclic voltammetry, and electrochemical impedance spectroscopy (EIS). The last technique was explored in order to check the adsorption of CT DNA onto the multilayer porphyrin/quantum dot assembly. The difference in electron transfer resistance (ΔR ct) obtained after CT DNA incubations showed the best result for a specific multilayer assembly, n = 3. {[Ttolyl(P-(C6H5)3)4]4+/CdTe}3 showed a good correlation of ΔR ct with the logarithmic concentration of CT DNA, in the range 1.0 × 10−10 to 1.0 × 10−6 M with a limit of detection (LOD) of 1.5 × 10−12 M. Conversely, when the {[Ttolyl(P-(C6H5)3)4]4+/CdTe}3 system was incubated with calf thymus single-stranded DNA and salmon testes DNA, no significant difference in ΔR ct was observed. We conclude that the newly described {[Ttolyl(P-(C6H5)3)4]4+/CdTe} n system is a choice method for the impedimetric determination of CT DNA.

Quantification of neurotransmitters and metabolically related compounds at glassy carbon electrodes modified with bamboo-like carbon nanotubes dispersed in double stranded DNA

This work reports the application of glassy carbon electrodes (GCE) modified with bamboo-like carbon nanotubes (bCNT) dispersed in calf-thymus double-stranded DNA (dsDNA) (GCE/bCNT-dsDNA) for the highly sensitive and selective quantification of neurotransmitters and metabolically related compounds by Adsorptive Stripping. Dopamine (Do) was quantified in the presence of its most important metabolite, 3,4-dihydroxyphenylacetic acid (dopac), while normetanephrine (NM) was determined in the presence of its precursor norepinephrine (NE). The accumulation of the analytes was performed at open circuit potential and the quantification was carried out, after medium exchange, in a 0.200M acetate buffer solution pH5.0 using Differential Pulse Voltammetry. The sensitivity for Do in the presence of 1.0×10−5M dopac was (7.1±0.2)×105μAM−1, (just 7.0% higher than the one obtained for Do alone), the detection limit was 68nM, and the linear range 5.0×10−7M-7.0×10−6M. The sensitivities for NM in the absence and presence of NE were (2.8±0.1)×104μAM−1 and (2.6±0.2)×104μAM−1, with a linear range between 1.0×10−6 and 2.0×10−5M, and a detection limit of 0.5μM. The platform was successfully used for the quantification of Do and NM in enriched human urine samples.

Spectroscopic, electrochemical, DNA binding and antioxidant biomimetic catalytic activities of metformin-based copper(II) complexes

Interaction of copper(II) perchorate hexahydrate with metformin as a primary ligand and amino acids as secondary ligands lead to the formation of three mixed ligand complexes, viz., [Cu(Met)(AA)(ClO4)(H2O)] (Met=metformin, AA=glycine (1), L-glutamine (2), and L-arginine (3)). Their structures and properties were characterized by elemental analysis, IR, UV–Vis, and ESR spectroscopy, electrochemical measurements including cyclic voltammetry, electrical molar conductivity and magnetic moment measurements. An octahedral geometry is proposed for the three compounds in monomeric structures. The redox behavior of complexes was investigated by cyclic voltammetry. Also, the interaction of copper complexes with calf thymus double-stranded DNA (CT-DNA) was studied electrochemically by using CV. The binding constants of complexes (2) and (3) with DNA were determined through the changes on the redox peak currents. The binding constants were evaluated to be 4.6×104 and 2.8×104M−1 for complexes (2) and (3), respectively. Additionally, the antioxidant (superoxide dismutase and catalase) biomimetic catalytic activities of the obtained complexes (1–3) have been assessed for their efficiency of O2− scavenging with and complete reactive oxygen species (ROS) detoxification with respect to peroxides.

Reversibility of the interactions between a novel surfactant derived from lysine and biomolecules

In this work the novel cationic surfactant derived from lysine (S)-5-acetamido-6-(dodecylamino)-N,N,N-trimethyl-6-oxohexan-1-ammonium chloride, LYCl, was prepared and the physicochemical characterization of its aqueous solutions was carried out. The binding of LYCl to bovine serum albumin, BSA, and to double stranded calf thymus DNA, ctDNA, was investigated using several techniques. Results show that LYCl binding to BSA is followed by a decrease in the α-helix content caused by the unfolding of the protein. LYCl association to ctDNA mainly occurs through groove binding and electrostatic interactions. These interactions cause morphological changes in the polynucleotide from an elongated coil structure to a more compact globular structure, resulting in the compaction of ctDNA. Addition of β-cyclodextrin, β-CD, to the BSA–LYCl and ctDNA–LYCl complexes is followed by the refolding of BSA and the decompaction of ctDNA. This can be explained by the ability of β-CD to hinder BSA–LYCl and ctDNA–LYCl interactions due to the stronger and more specific β-CD–LYCl hydrophobic interactions. The stoichiometry of the β-CD:LYCl inclusion complex and its formation equilibrium constant were determined in this work. The reported procedure using β-CD is an efficient way to refold proteins and to decompact DNA, after the morphological changes caused in the biomolecules by their interaction with cationic surfactants.

Determination of guanine and adenine by high-performance liquid chromatography with a self-fabricated wall-jet/thin-layer electrochemical detector at a glassy carbon electrode

A sensitive wall-jet/thin-layer amperometric electrochemical detector (ECD) coupled to high-performance liquid chromatography (HPLC) was developed for simultaneous determination of guanine (G) and adenine (A). The analytes were detected at a glassy carbon electrode (GCE) and the HPLC−ECD calibration curves showed good linearity (R2>0.997) under optimized conditions. Limits of detection for G and A are 0.6nM and 1.4nM (S/N=3), respectively, which are lower than those obtained with an UV–vis detector and a commercial electrochemical detector. We have successfully applied this HPLC−ECD to assess the contents of G and A in hydrochloric acid-digested calf thymus double-stranded DNA. In addition, we compared in detail the analysis of G and A by cyclic voltammetry (CV) and by the HPLC−ECD system on both bare GCE and electroreduced graphene oxide (ERGO) modified GCE. We found that the adsorption of G and A on the electrode surfaces can vary their anodic CV peaks and the competitive adsorption of G and A on the limited sites of the electrode surfaces can cause crosstalk effects on their anodic CV peak signals, but the HPLC−ECD system is insensitive to such electrode-adsorption and can give more reliable analytical results.

Disposable electrochemical DNA biosensor for environmental monitoring of toxicant 2-aminoanthracene in the presence of chlorine in real samples

A simple procedure for the voltammetric detection of the DNA damage using a disposable electrochemical DNA biosensor is reported. The DNA biosensor is assembled by immobilizing the double stranded calf thymus DNA (dsDNA) on the surface of a disposable carbon screen-printed electrode. The interaction of 2-aminoanthracene (2-AA) with calf thymus dsDNA was studied electrochemically based on the oxidation signals of guanine (G) and adenine (A) by using square wave voltammetry (SWV) at screen printed electrode (SPE). The oxidation signals of the guanine and adenine bases, obtained by a square wave voltammetric scan, were used as analytical signal to detect the DNA damage. The presence of this aromatic amine compound with affinity for nucleic acids was measured by its effect on the guanine and adenine oxidation peaks. The response was obtained in the range of 0.05–20 mg L−1 for 2-AA concentration on dsDNA-modified SPE. This test has been used due to its rapid, easy handling and cost effective responses for the toxicity assessment in real water and bleach solution samples. A low-cost screen-printed DNA biosensor was developed as a simple strategy for the voltammetric detection of the DNA damage. The natural electrochemistry of guanine and adenine bases was exploited to develop the DNA-based biosensor.

Multivariate analysis for resolving interactions of carbidopa with dsDNA at a fullerene-C60/GCE

For the first time, interactions of carbidopa (CD) with double-stranded calf thymus DNA (dsDNA) in a phosphate buffered solution (PBS, 0.05M, pH=4.0) at a fullerene-C60/glassy carbon electrode (FLR/GCE) has been studied by cyclic voltammetry (CV), linear sweep voltammetry (LSV), and square wave voltammetry (SWV). The interaction of CD with dsDNA was also monitored using fluorescence (F) and UV–vis spectroscopic techniques. New information was obtained when a row- and column-wise augmented matrix consisting of SWV, LSV, F and UV–vis data was resolved using multivariate curve resolution-alternating least squares (MCR-ALS) as a powerful chemometric tool. Pure electrochemical and spectroscopic signals of CD, dsDNA and dsDNA–CD2 complex, and their concentration profiles were then successfully resolved. Molecular docking studies confirmed that the binding of CD with dsDNA shows minor groove binding mode which was in accordance with experimental results. Under optimized conditions, the SWV responses were linearly related to dsDNA concentration between 0.1 and 25.0nM and a limit of detection (LOD) of 0.03nM was calculated (3Sb/b=3). Moreover, the modified electrode exhibited long term stability, good repeatability, and reproducibility, and high sensitivity and selectivity toward dsDNA determination in human serum samples, demonstrating its feasibility toward dsDNA sensing.

An electrochemical chiral sensing platform for propranolol enantiomers based on size-controlled gold nanocomposite

The size-controlled gold nanoparticle–methylene blue–multiwalled carbon nanotubes nanocomposite (nanoAu–MB–MWNTs) was successfully synthesized as electrochemical redox-probe indicator and immobilization matrix, which calf thymus double-stranded DNA (ctDNA) could be adsorbed for chiral sensing propranolol (PRO) enantiomers. Scanning electron microscopy (SEM), transmission electron microscopy (TEM) and ultraviolet–visible spectroscopy (UV–vis) were used to characterize the functionalized nanocomposite. The DNA-immobilized sensing platform (ctDNA/nanoAu–MB–MWNTs/GCE) showed a larger electrochemical response for S-PRO than R-PRO, in which the association constant (β) was calculated to be 1.154×104Lmol−1 for S-PRO and 4.638×103Lmol−1 for R-PRO. Quartz crystal microbalance (QCM) and electrochemical impedance spectroscopy (EIS) had confirmed the higher affinity for S-PRO with ctDNA. The experimental parameters such as acidity, incubation time, size of nanoAu and the amount of ctDNA were assessed to obtain the optimized conditions. Moreover, the proposed sensor was applied to determine the enantiomeric ratios of R-PRO in mixture solutions and high-performance liquid chromatography (HPLC) was used to validate the results by calculating F- and t-test values at 95% confidence level.

Adsorptive transfer voltammetry applied to the study of chromium-induced DNA damage in the presence of curcumin

The interaction between double-stranded (ds) calf-thymus DNA and chromium in the presence of curcumin (CC) was studied by differential pulse adsorptive transfer voltammetry using carbon paste electrode (CPE). Curcumin–Cr complex generated changes in calf thymus DNA. The mechanism for DNA cleavage by curcumin–Cr complex appears to involve both the hydroxyl radical as well as singlet oxygen. The characteristic peak of dsDNA, due to the oxidation of guanine residues, drastically decreased. The increased DNA damage by curcumin–Cr complex was observed in the presence of various concentrations of chromium(VI).

Electrochemical Investigation and DNA-Binding Studies of Valacyclovir-Copper(II) Complex

The synthesis and the electrochemical characterization of copper(II) complex containing one molecule of antiviral drug, valacyclovir (VAL), is reported. In addition to characterization, the electrochemical properties of the copper(II) complex of VAL ([Cu(VAL)Cl2]) were investigated by cyclic voltammetry (CV) using glassy carbon electrode. The redox properties of the metal complex was reversible and exhibited diffusion controlled process depending on pH. The dependence of intensities of currents and potentials on pH, concentration, scan rate, and nature of the buffer was investigated. The oxidation/reduction mechanism was proposed and discussed. Also, the interaction of copper complex of VAL- with calf thymus double-stranded DNA was studied spectrophotometrically (UV-vis) and electrochemically by using CV at glassy carbon working electrode. Additionally, the cytotoxicity activity of complex has been investigated against the bovine herpes virus type I.

Electrochemical study of dsDNA on carbon nanotubes paste electrodes applying cyclic and differential pulse voltammetry

Abstract Abstract Carbon nanotubes paste electrodes (CNTPEs) in combination with adsorptive transfer stripping voltammetry are shown to be very suitable for the determination of calf thymus double-stranded DNA (dsDNA). The performance of three types of multi-walled carbon nanotubes paste electrodes (MWCNTPEs) is investigated. The effects of surface pre-treatment and accumulation conditions on the adsorption and electrooxidation of the dsDNA at MWCNTPEs are also described. The results indicate that the electroactivity inherent to carbon nanotubes/paste electrodes allows a large enhancement of the guanine oxidation signal compared to that obtained at the conventional carbon paste electrodes (CPEs). Moreover, the extent of the enhancement dependents on the type of MWCNTs incorporated into the paste. Based on the signal of guanine, under optimal conditions, very low levels of dsDNA can be detected following short accumulation times for all three types of MWCNTPEs (MWCNTPE1, MWCNTPE2, MWCNTPE3), with detection limits of 2.64 mg L−1, 2.02 mg L−1 and 1.46 mg L−1, respectively. Additionally, the dsDNA isolated from rat liver tissues is determined by use of the previously mentioned MWCNTPEs. Graphical abstract

Electrochemical DNA Sensors for Rapid Detection of DNA Damage Induced by Fenton Reaction

Abstract A DNA electrochemical sensor was prepared by assembling layer-by-layer films of poly dimethyl diallyl ammonium chloride and calf thymus double-stranded DNA (CT-dsDNA) on the surface of a glassy carbon electrode (GCE). The assembling DNA was characterized by electrochemical methods and atomic force microscope. Ru(bpy) 3 2+ was used as electrochemical catalyst to detect the oxidative damage of DNA in the membrane. The effects of incubation time and concentration of Fenton reagent on the damage of DNA were studied. The result showed that it could cause damage greatly when incubated in Fenton reagent for 15 min. The detectable concentration of Fenton reagent was 5.0 × 10 −5 M FeSO 4 /2.0 × 10 −4 M H 2 O 2 . The electrochemical method possesses high-sensitivity for detecting the damage of DNA.

Interaction of sulfadiazine with DNA on a MWCNT modified glassy carbon electrode: Determination of DNA

The interactions of sulfadiazine (SD), an antimicrobial drug, with double-stranded calf thymus DNA on the multi-walled carbon nanotubes modified glassy carbon electrode (MWCNT-GCE) have been studied by cyclic voltammetry and UV–vis spectroscopy. In the presence of DNA, the oxidation peak current of SD decreases and the peak potential shifts to a positive potential which indicates the interaction of SD with DNA. The binding of SD with DNA shows both electrostatic and intercalative modes. The binding of SD with DNA, when analyzed in terms of the cooperative Hill model, yields the binding constant, Ka=2.87×103M−1 and a Hill coefficient m=1.9 in Britton–Robinson (B-R) buffer solution, pH=6.5. This electrochemical method was further applied to the determination of DNA. Under the selected conditions, two linear calibration curves for DNA detection were obtained in the concentration ranges from 0.03 to 0.13μgmL−1 and 0.60–3.50μgmL−1 with detection limit 0.03μgmL−1. The method was also applied to the determination of DNA in human blood plasma sample.

Quantification of 8-OxodGuo Lesions in Double-Stranded DNA Using a Photoelectrochemical DNA Sensor

Exposure of DNA to oxidative stress conditions results in the generation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo). 8-OxodGuo is genotoxic if left unrepaired. We quantified 8-oxodGuo lesions in double-stranded DNA films by using a photoelectrochemical DNA sensor in conjunction with a specific covalent labeling method. A lesion-containing DNA film was assembled on a SnO(2) nanoparticle modified indium tin oxide electrode through layer-by-layer electrostatic adsorption. The lesions were covalently labeled with a biotin conjugated spermine derivative, and ruthenium tris(bipyridine) labeled streptavidin was introduced as the signal reporter molecule. Photocurrent increased with the number of lesions in the strand and decreased as the film was diluted with intact DNA. Quantification of 8-oxodGuo was achieved with an estimated detection limit of ∼1 lesion in 650 bases or 1.6 fmol of 8-oxodGuo on the electrode. Incubation of the film with a DNA base excision repair enzyme, E. coli formamidopyrimidine-DNA glycosylase (Fpg), resulted in complete loss of the signal, indicating efficient excision of the isolated lesions in the nucleotide. Oxidatively generated DNA damage to a double-stranded calf thymus DNA film by the Fenton reaction was then assessed. One 8-oxodGuo lesion in 520 bases was detected in DNA exposed to 50 μM Fe(2+)/200 μM H(2)O(2). Treatment with Fpg reduced the photocurrent by 50%, indicating only partial excision of 8-oxodGuo. This suggests that tandem lesions, which are resistant to Fpg excision, are generated by the Fenton reaction. Unlike repair enzyme dependent methods, the sensor recognizes 8-oxodGuo in tandem lesions and can avoid underestimating DNA damage.

Investigação eletroquímica e calorimétrica da interação de novos agentes antitumorais biscatiônicos com DNA

Biscationic amidines bind in the DNA minor groove and present biological activity against a range of infectious diseases. Two new biscationic compounds (bis-α,ω-S-thioureido, amino and sulfide analogues) were synthesized in good yields and fully characterized, and their interaction with DNA was also investigated. Isothermal titration calorimetry (ITC) was used to measure the thermodynamic properties of binding interactions between DNA and these ligands. A double stranded calf thymus DNA immobilized on an electrode surface was used to study the possible DNA-interacting abilities of these compounds towards dsDNA in situ. A remarkable interaction of these compounds with DNA was demonstrated and their potential application as anticancer agents was furthered.

Voltammetric DNA Biosensor Based on a Microcrystalline Natural Graphite–Polystyrene Composite Transducer

New voltammetric DNA biosensor based on a microcrystalline natural graphite–polystyrene composite film in the role of a transducer was used for the investigation of the interaction between model carcinogenic substance (2-aminofluorene; one of the most extensively studied examples of the aromatic amine class of carcinogens) and calf thymus double-stranded DNA (dsDNA). The layer of dsDNA immobilized at the electrode surface was utilized as a biocomponent responsive interface. The biosensor was characterized regarding the detection of DNA damage (induced by direct interaction with 2-aminofluorene) using square wave voltammetric responses of the guanine and adenine moieties and cyclic voltammetric responses of the anionic redox indicator [Fe(CN)6]4–/3– present in the solution.

Ribbon-type and cluster-type lipoplexes constituted by a chiral lysine based cationic gemini lipid and plasmid DNA

Lipoplexes constituted by plasmid DNA pEGFP-C3 (pDNA) or linear double-stranded calf thymus DNA (ctDNA) and mixed cationic liposomes consisting of several percentages of the cationic lysine derived lipid C6(LL)2 and the zwitterionic lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) have been analyzed by both experimental and theoretical approaches. Experimental studies, consisting of electrophoretic mobility/zeta potential, small angle X-ray scattering (SAXS), cryogenic transmission electron microscopy (cryo-TEM), negatively stained transmission electron microscopy (NS-TEM), and GelRed f1uorescence intercalation assays, have been carried out at several liposome and lipoplex compositions, defined in terms of cationic lipid molar fraction and either the mass or charge ratios of the lipoplex. The electrochemical study confirms that, in the presence of the mixed lipids and in contrast with what has usually been found for linear DNA, the plasmid DNA is compacted with a large number of its Na+ counterions, thus yielding a much lower effective negative charge (q−pDNA) than that for ctDNA (q−ctDNA), as reported recently by us (J. Am. Chem. Soc., 2011) for other lipoplexes. This finding is revealed as crucial for an optimum and efficient lipoplex preparation, since a lower effective negative charge implies a lower quantity of cationic lipid and, accordingly, a potential lower cytotoxicity. TEM experiments reveal a complex scenario of multilamellar nanostructures, from ribbon-type (typically present for chiral lipids) to cluster-type structures (usually found in cationic lipid/DOPE systems), the composition of the mixed liposome playing an important role in the final morphology of the lipoplex. SAXS diffractograms confirm the existence of these two types of multilamellar structures through a deconvolution process of the first peak of diffractograms into two overlapping bands. On the other hand, a theoretical complexation model is employed to determine the net charge of the lipoplexes studied in this work. The model allows analysis and comparison of the electrochemical behaviour of lipoplexes containing linear DNA vs. those constituted by a supercoiled DNA, confirming the experimental findings.

Calf thymus DNA-stabilized polythiophene fluorescence probe for label-free detection of spermine

It is a challenge to detect molecules lacking a chromophore, such as polyamines, by optical methods since they are insensitive to light. In order to detect the optical signals, it is compulsory to derive these molecules with optical labels, which, however, is complicated, time-consuming and may be expensive. In this work, a highly specific strategy for spermine detection is developed on the basis of the iodide-induced conformational change of polythiophene. By using the triplex complex of negatively charged double-stranded calf thymus DNA (ctDNA)-stabilized cationic polythiophene as a fluorescent probe, the highly specific detection of spermine could be realized since polythiophene, which will be released from the triplex complex owing to the condensation and aggregation of ctDNA with spermine, undergoes a conformational change from the random-coiled non-planar state to the highly conjugated planar form in the presence of iodide, resulting in a yellow-to-red color conversion and fluorescence quenching. The quenched fluorescence was found to be proportional to the spermine concentrations in the range of 1.2-50 μM with the limit of detection (LOD) being 0.5 μM (3σ/k).

Electrochemical probe of natural DNA attached onto the gold–thiol SAMs via Zr(IV) ion glue

Attachment of natural double strand calf thymus DNA onto gold–mercaptopropionic acid self-assembled monolayer electrode via Zr(IV) ion glue, Au–MPA–Zr(IV)-ds-ct-DNA SAM, is studied. The sequential assembly of the layers on the Au surface is traced by electrochemical methods including cyclic and square wave voltammetries in the presence of methylene blue. The results show that DNA is attached to Zr(IV) via its phosphate groups without any contribution from its bases. Effects of the MB concentration on the behavior of adsorbed MB, ionic strength on the MB adsorption, and pH on the amount of immobilized DNA are studied. The proposed method for the preparation of the new template, the Au–MPA–Zr(IV)-ds-ct-DNA, is relatively simple and fast. Long-term stability of this electrode is tested by keeping the template at pH 3, and carrying out a daily electrochemical study at pH 7.4. Under these circumstances, the Au–MPA–Zr(IV)-ds-ct-DNA electrode showed stable behavior for one week.

DNA immobilization on a polypyrrole nanofiber modified electrode and its interaction with salicylic acid/aspirin

A double-stranded calf thymus DNA (dsDNA) was physisorbed onto a polypyrrole (PPy) nanofiber film that had been electrochemically deposited onto a Pt electrode. The surface morphology of the polymeric film was characterized using scanning electron microscopy (SEM). The electrochemical characteristics of the PPy film and the DNA deposited onto the PPy modified electrode were investigated by cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS). Then the interaction of DNA with salicylic acid (SA) and acetylsalicylic acid (ASA), or aspirin, was studied on the electrode surface with DPV. An increase in the DPV current was observed due to the oxidation of guanine, which decreased with the increasing concentrations of the ligands. The interactions of SA and ASA with the DNA follow the saturation isotherm behavior. The binding constants of these interactions were 1.15×10(4)M for SA and 7.46×10(5)M for ASA. The numbers of binding sites of SA and ASA on DNA were approximately 0.8 and 0.6, respectively. The linear dynamic ranges of the sensors were 0.1-2μM (r(2)=0.996) and 0.05-1mM (r(2)=0.996) with limits of detection of 8.62×10(-1) and 5.24×10(-6)μM for SA and ASA, respectively.