Electrochemical DNA biosensor for the study of ciprofloxacin-DNA interaction

The interaction of ciprofloxacin with DNA was studied by using an electrochemical DNA biosensor. The binding mechanism of ciprofloxacin was elucidated by using constant current potentiometry and differential pulse voltammetry at DNA-modified glassy carbon electrode. The decrease in the guanine oxidation peak area or peak current at +0.9 V was used as an indicator for the interaction mechanism in 0.2M acetate buffer (pH 5). The binding constant (K) values obtained were 1.33+/-0.02 x 10(4) and 1.32+/-0.08 x 10(4) M(-1) with constant current potentiometry and differential pulse voltammetry, respectively. A linear dependence of the guanine peak area or peak currents was observed in the range of 40-80 microM ciprofloxacin, with a detection limit of 24 microM with r=0.995 and 9 microM with r=0.999 by using constant current potentiometry and differential pulse voltammetry, respectively. Moreover, the influence of sodium and calcium ions was also studied to elucidate the mechanism of ciprofloxacin-DNA interaction at different solution conditions, and this proved to be helpful in understanding the ciprofloxacin-DNA interaction.     

Electrochemically fabricated polypyrrole nanofiber-modified electrode as a new electrochemical DNA biosensor

A new biosensor employing immobilized DNA on a nano-structured conductive polymer fixed onto a platinum electrode is presented. Upon optimization of synthesis parameters, polypyrrole nanofibers, 30-90 nm in diameter, were synthesized in an aqueous media by the electropolymerization of pyrrole using normal pulse voltammetry (NPV). The nanofiber film was investigated by scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Double-stranded DNA was physisorbed onto the PPy nanofiber films. Various parameters, including the pH and DNA concentration, were optimized. The DNA immobilized on the nanofiber films was characterized using differential pulse voltammetry (DPV) and Fourier-transform infrared (FTIR) spectroscopy. Using DPV to study the interaction of spermidine with DNA, a binding constant (K) value of 4.08 x 10(5)+/-0.05 M(-1) was obtained. For the determination of spermidine, the proposed method exhibited a good dynamic range, correlation coefficient (0.05-1.0 microM and 0.9983, respectively) and a low detection limit (0.02 microM), although Ca(2+) ions were found to electrostatically bind to DNA and weaken the spermidine-DNA interaction.     

Binding of oxovanadium ions to the major and minor grooves of DNA duplex: stability and structural models.

Vanadate induces DNA strand breaks in cultured human fibroblasts at doses that are relative to the occupational exposure. Oxovanadium compounds also exert preventive effects against chemical carcinogenesis in animals and form complexes with DNA in vivo. This study was designed to examine the interaction of calf-thymus DNA with VO2+ and VO3 ions in aqueous solution at physiological pH, with a constant DNA concentration of 12.5 mmol/L and vanadium-DNA (phosphate) molar ratios (r) of 1:160 to 1:2. Capillary electrophoresis and Fourier transform infrared difference spectroscopy were used to determine the cation binding site, the binding constant, the helix stability, and DNA conformation in the oxovanadium-DNA complexes. Structural analysis showed that VO2+ binds DNA through guanine and adenine N-7 atoms and the backbone PO2 group with apparent binding constants of KG = 8.8 x 10(5) (mol/L)-1 and KA = 3.4 x 10(5) (mol/L)-1. The VO3 shows weaker binding through thymine, adenine, and guanine bases, with K = 1.9 x 10(4) (mol/L)-1 and no interaction with the backbone phosphate group. A partial B-to-A DNA transition occurred upon VO-DNA complexation, while DNA remains in the B-family structure in the VO3 complexes.     

Thymol and carvacrol binding to DNA: model for drug-DNA interaction

Thymol and carvacrol can bind to major and minor grooves of B-DNA. The aim of this study was to examine the interaction of calf thymus DNA with thymol and carvacrol in aqueous solution and physiological pH with thymol/DNA and carvacrol/DNA (phosphate) molar ratios of 1/20, 1/10, 1/5, and 1/1. Fourier transform infrared and UV-visible difference spectroscopy were used to determine the thymol and carvacrol binding mode, binding constant, sequence selectivity, DNA secondary structure, and structural variations of thymol/DNA and carvacrol/DNA complexes in aqueous solution. Spectroscopic evidence showed that the thymol and carvacrol interaction occurred mainly through H-bonding of the thymol and carvacrol OH group to the guanine N7, cytosine N3, and backbone phosphate group with overall binding constant of K(thymol-DNA) = 2.43 x 10(3) M(-1), K(carvacrol-DNA) = 1.55 x 10(3) M(-1). In thymol and carvacrol-DNA complexes, DNA remains in the B-family structure.     

Binding of ellipticine base and ellipticinium cation to calf-thymus DNA. A thermodynamic and kinetic study

The acid-basic properties of ellipticine have been re-estimated. The apparent pK of protonation at 3 microM drug concentration is 7.4 +/- 0.1. The ellipticine free base (at pH 9, I = 25 mM) intercalates into calf-thymus DNA with an affinity constant of 3.3 +/- 0.2 X 10(5) M-1, and a number of binding sites per phosphate of 0.23. The ellipticinium cation (pH 5, I = 25 mM) binds also to DNA with a constant of 8.3 +/- 0.2 x 10(5) M-1 and at a number of binding sites (n = 0.19). It is postulated that the binding of the drug to DNA at pH 9 is driven by hydrophobic and/or dipolar effects. Even at pH 5, where ellipticine exists as a cation, it is thought that the hydrophobic interaction is the main contribution to binding. The neutral and cationic forms share common binding within DNA sites but yield to structurally different complexes. The free base has 0.04 additional specific binding sites per phosphate. As determined from temperature-jump experiments, the second-order rate constant of the binding of the free base (pH 9) is 3.4 x 10(7) M-1 s-1 and the residence time of the base within the DNA is 8 ms. The rate constant for the binding of the ellipticinium cation is 9.8 x 10(7) M-1 s-1 when it is assumed that drug attachment occurs via a pathway in which the formation of an intermediate ionic complex is not involved (competitive pathway).     

Study of interactions between DNA and aflatoxin B1 using electrochemical and fluorescence methods

In this study, a carbon paste electrode modified with N-butylpyridinium hexafluorophosphate (BPPF₆) ionic liquid and DNA was introduced as an electrochemical biosensor to study the interaction between DNA and aflatoxin B1 molecules. For this purpose, variations in oxidation peak current of guanine in various concentrations of aflatoxin B1 were measured by using the differential pulse voltammetry (DPV) method. According to this study, the binding constant of DNA–aflatoxin B1 was found to be 3.5 × 10⁶ M⁻¹. This modified electrode was also used for determination of low concentrations of aflatoxin B1 by using differential pulse voltammetry. A linear dynamic range from 8.00 × 10⁻⁸ to 5.91 × 10⁻⁷ M and a limit of detection of 2.00 × 10⁻⁸ M resulted from DPV measurements. To confirm our results, a fluorescence study was also performed. It resulted in a binding constant of 2.8 × 10⁶ M⁻¹, which is in good agreement with that obtained from electrochemical study.     

The interaction of ribonuclease T 1 with DNA.

The interaction of RNase T1 with calf thymus DNA was studied using uv difference spectroscopy and the effect of the enzyme on DNA melting. There was no indication of RNase T1 binding with native DNA. A prominent difference spectrum for RNase T1 binding with denatured DNA (d-DNA) was observed at pH 5, 25 degrees and low ionic strength (mu = .01 M) which was depressed at higher ionic strength and pH. The normalized difference spectrum at mu = .01 M, pH 5 and 25 degrees can be interpreted as indicating an interaction of an exposed guanine residue directly with the enzyme and a coupling of this process with the "melting" of short folded segments of d-DNA. The apparent association constant calculated per M guanine residues was 2.4 X 10-4 M-1 under these conditions. The results are discussed in reference to comparable studies on the interaction of RNase T1 with RNA and small guanine ligands.     

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

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

Study on the interaction of bovine serum albumin with acid cyanine 5R and its application in analysis.

A supramolecular complex of bovine serum albumin (BSA) with acid cyanine 5R (AC 5R, C.I. acid blue 113, C.I.: 26360) has been shown to form in Tris-HCl buffer solution (pH 7.42) by linear sweep voltammetry (LSV), fluorimetry, and spectrophotometry. The binding ratio and binding constant of BSA with AC 5R have been detected by LSV and fluorimetry. The binding mechanism is also preliminarily discussed. In Tris-HCl buffer solution (pH 7.42), AC 5R can easily be reduced on the mercury electrode, and it has a well-defined LSV peak current (Ip) and peak potential (Ep) at -0.65 V (vs. SCE). In the presence of BSA, the Ip of AC 5R decreases, and the peak potential (Ep) shifts to a more positive potential. The decrease of the second-order derivative of reductive peak current (deltaIp') of AC 5R is proportional to the logarithm of BSA concentration in the range of 1.54 x 10(-8) mol x L(-1)-1.54 x 10(-5) mol x L(-1) (r = 0.9931-0.9977). The limit of detection of BSA is 9.0 x 10(-9) mol x L(-1). The relative standard deviation is 1.83% (n = 10), and the standard recovery is 97.5%-104.8%. This method can be used to determine BSA concentration on the basis of the interaction of BSA with AC 5R.     

A Comparative study of Fe(II) and Fe(III) interactions with DNA duplex: major and minor grooves bindings

The involvement of the Fe cations in autoxidation in cells and tissues is well documented. DNA is a major target in such reaction, and can chelate Fe cation in many ways. The present study was designed to examine the interaction of calf-thymus DNA with Fe(II) and Fe(III), in aqueous solution at pH 6.5 with cation/DNA (P) (P = phosphate) molar ratios (r) of 1:160 to 1:2. Capillary electrophoresis and Fourier transform infrared (FTIR) difference spectroscopic methods were used to determine the cation binding site, the binding constant, helix stability and DNA conformation in Fe-DNA complexes. Structural analysis showed that at low cation concentration (r = 1/80 and 1/40), Fe(II) binds DNA through guanine N-7 and the backbone PO(2) group with specific binding constants of K(G) = 5.40 x 10(4) M(1) and K(P) = 2.40 x 10(4) M(1). At higher cation content, Fe(II) bindings to adenine N-7 and thymine O-2 are included. The Fe(III) cation shows stronger interaction with DNA bases and the backbone phosphate group. At low cation concentration (r = 1:80), Fe(III) binds mainly to the backbone phosphate group, while at higher metal ion content, cation binding to both guanine N-7 atom and the backbone phosphate group is prevailing with specific binding constants of K(G) = 1.36 x 10(5) M(-1) and K(P) = 5.50 x 10(4) M(-1). At r = 1:10, Fe(II) binding causes a minor helix destabilization, whereas Fe(III) induces DNA condensation. No major DNA conformational changes occurred upon iron complexation and DNA remains in the B-family structure.     

Voltammetric behaviour of DNA bases at a screen-printed carbon electrode and its application to a simple and rapid voltammetric method for the determination of oxidative damage in double stranded DNA

Screen-printed carbon electrodes (SPCEs) have been investigated as possible sensors to identify gamma-irradiation induced oxidative damage in double stranded (ds) DNA. Studies were undertaken to explore the possibility of using both cyclic voltammetry and differential pulse voltammetry to identify changes due to oxidative damage. Initially, guanine, adenine and 8-oxoguanosine were examined and it was found possible to differentiate them from their voltammetric responses. The voltammetric response of 8-oxoguanosine was found to be linear over the concentration range 1-400 microM, with a slope of 0.0296 microA microM(-1) (R2 value of 0.9984), in the presence of 2mM concentrations of guanine and adenine. Investigations were made into harnessing these findings to identify oxidative damage in gamma-irradiated dsDNA. The presence of oxidative damage in these samples was readily identifiable, and the magnitude of the voltammetric response was found to be dose dependant (R2=0.9919). A simple sample preparation step involving only the dissolution of double stranded DNA sample in the optimised electrolyte (0.1M acetate buffer pH 4.5) was required. This report appears to be first describing the use of a SPCE to detect DNA damage which can be related to the dose of gamma-radiation used.     

Cleavage of DNA by electrochemically activated MnIII and FeIII complexes of meso-tetrakis (N-methyl-4-pyridiniumyl)porphine

Electrochemical methods were used to activate MnIII and FeIII complexes of meso-tetrakis(N-methyl-4-pyridiniumyl)porphine (H2TMPyP) to cause cleavage of pBR322 DNA and to study their interaction with sonicated calf thymus DNA. Electrochemical reduction of MnIIITMPyP and FeIIITMPyP (at low concentrations) in the presence of O2 was required to activate these complexes. However, FeIIITMPyP at 1 x 10(-6) M produced DNA strand breakage without being electrochemically reduced. At low concentrations, FeIITMPyP was more efficient at cleaving DNA than MnIITMPyP. Reduction of O2 at a platinum electrode also produced some cleavage but to a much smaller extent. The oxidized form of MnIIITMPyP (charge 5+) has higher affinity for sonicated calf thymus (CT) DNA than the reduced form (charge 4+), as determined by the negative shift in E degrees' for the voltammetric wave in the presence of DNA. Both forms of FeIIITMPyP (charge 4+) interact with DNA to about the same extent. Differential pulse voltammetry was used to determine binding constants (K) and binding-site sizes (s) of the interaction of these metalloporphyrins with sonicated CT DNA. The data were analyzed assuming both mobile and static equilibria. MnIIITMPyP binds to DNA (5 mM Tris, 50 mM NaCl, pH 7) with K = 5 (+/- 2) x 10(6) M-1, s = 3 bp (mobile) or K = 3.6 (+/- 0.3) x 10(6) M-1, s = 4 bp (static). FeIIITMPyP at that ionic strength caused DNA precipitation. At higher ionic strength (0.1 M Tris, 0.1 M NaCl, pH 7), FeIIITMPyP associates to DNA with K = 4.4 (+/- 0.2) x 10(4) M-1, s = 5 bp (mobile) or K = 1.9 (+/- 0.1) x 10(4) M-1, s = 6 bp (static).     

One-pot solvent free synthesis and DNA binding studies of thieno[2,3-b]-1,8-naphthyridines

With the aim of evaluating interaction between double-stranded calf thymus (ds)DNA and sulphur containing fused planar rings, the derivatives of 1,8-naphthyridine containing thiono groups were synthesized by the condensation of 2-mercapto-3-formyl[1,8]naphthyridines using 1-chloroacetone, 2-chloroacetamide, chloroaceticacid, and 2-chloro-1-phenylethanone in the presence of anhydrous potassium carbonate as s catalyst under solvent free microwave irradiation. The structures of the compounds were elucidated on the basis of elemental analysis, IR, (1)H NMR, and mass spectra. The interaction of thieno[2,3-b]-1,8-naphthyridine-2-carboxylic acid (TNC) (3a) with ct-DNA was studied by UV-Vis spectrophotometry, viscosity, thermal denaturation, as well as cyclic voltammetry experiments. On binding to DNA, the absorption spectrum underwent bathochromic and hypochromic shifts. Binding parameters, determined from spectrophotometric measurements indicated a binding constant of Kb=2.1 x 10(6) M(-1). The thieno[2,3-b]-1,8-naphthyridine-2-carboxylic acid (3a) increases the viscosity of sonicated rod-like DNA fragments. The binding of TNC to DNA increased the melting temperature by about 4 degrees C. The decrease in peak current heights and shifts of peak potential values are observed by the addition of calf thymus DNA in cyclic voltammetry studies.     

Electrochemical determination of interaction parameters for DNA and mitoxantrone in an irreversible redox process

Mitoxantrone (MXT), an anti-tumor antibiotic, shows irreversible electrochemical behavior at a waxed graphite electrode in a 0.05 M Tris-HCl buffer (pH 7.4) solution. The interaction between MXT and calf thymus DNA (ctDNA) in solution has been studied using cyclic voltammetry. An electrochemical equation suitable for examining the binding of irreversibly electroactive molecules to DNA is established. Determination of diffusion coefficients of both free and binding MXT (D(f), D(b)), the binding constant (K) and binding site size (s base pairs per molecule, bp) of MXT with DNA was performed on the basis of the equation. A nonlinear fit analysis of the experimental data yielded: D(f)=3.76 x 10(-5) cm(2)s(-1), D(b)=2.73 x 10(-7) cm(2)s(-1), K=8.7 x 10(9) cm(3)mol(-1), s=2.8 bp. The results demonstrate that MXT binds tightly to ctDNA and covers three base pairs. The anthraquinone of MXT, which is a planar heterocyclic ring, intercalates between the DNA's base pairs. The two aminoethylamino side-chains of the drug fit to the major groove reinforce the combination of MXT and DNA. The results show that MXT is a DNA intercalator with a high binding constant compared to those of other anthraquinones.     

DNA interaction with RNase A alters protein conformation

DNA-RNase H adducts were used for site specific cleavage of RNA and DNA-RNA duplexes, whereas nonspecific DNA interaction with ribonuclease A (RNase A) has been observed. The aim of this study was to examine the complexation of calf-thymus DNA with RNase A at physiological condition, using constant DNA concentration (12.5 mM) and various protein contents (1 microM to 270 microM). FTIR, UV-visible, and CD spectroscopic methods were used to analyse protein binding mode, the binding constant and the effects of nucleic acid-enzyme interaction on both DNA and protein conformations. Our structural analysis showed a strong RNase-PO2 binding and minor interaction with G-C bases with overall binding constant of K = 6.1 x 10(4) M(-1). The RNase-DNA interaction alters the protein secondary structure with a major reduction of the alpha-helix and increase of the beta-sheet and random structure, while DNA remains in the B-family structure.     

Investigation on the interaction of DNA and electroactive ligands using a rapid electrochemical method

A rapid method for investigation of the interaction of DNA and electroactive ligands based on an electrochemical equation for irreversible processes is presented. The binding constant (K) and the size of binding site (s) are simultaneously obtained from the dependence of the current on the amount of added DNA in voltammetry. A non-intercalative binder (Hoechst 33258) and two DNA-intercalators (mitoxantrone (MXT) and actinomycin D (AMD)) were examined in experiments. It was found that the binding constant of Hoechst 33258, mitoxantrone and actinomycin D, were 2.1 x 10(8), 8.9 x 10(9) and 9.1 x 10(9) cm(3) mol(-1); and the size of their binding sites were 4, 3 and 8, respectively. The study provides a convenient and sensitive approach for estimating affinity parameters and outlining the interaction between DNA and electroactive targeting compounds.     

Silver(I) complexes with DNA and RNA studied by Fourier transform infrared spectroscopy and capillary electrophoresis

Ag(I) is a strong nucleic acids binder and forms several complexes with DNA such as types I, II, and III. However, the details of the binding mode of silver(I) in the Ag-polynucleotides remains unknown. Therefore, it was of interest to examine the binding of Ag(I) with calf-thymus DNA and bakers yeast RNA in aqueous solutions at pH 7.1-6.6 with constant concentration of DNA or RNA and various concentrations of Ag(I). Fourier transform infrared spectroscopy and capillary electrophoresis were used to analyze the Ag(I) binding mode, the binding constant, and the polynucleotides' structural changes in the Ag-DNA and Ag-RNA complexes. The spectroscopic results showed that in the type I complex formed with DNA, Ag(I) binds to guanine N7 at low cation concentration (r = 1/80) and adenine N7 site at higher concentrations (r = 1/20 to 1/10), but not to the backbone phosphate group. At r = 1/2, type II complexes formed with DNA in which Ag(I) binds to the G-C and A-T base pairs. On the other hand, Ag(I) binds to the guanine N7 atom but not to the adenine and the backbone phosphate group in the Ag-RNA complexes. Although a minor alteration of the sugar-phosphate geometry was observed, DNA remained in the B-family structure, whereas RNA retained its A conformation. Scatchard analysis following capillary electrophoresis showed two binding sites for the Ag-DNA complexes with K(1) = 8.3 x 10(4) M(-1) for the guanine and K(2) = 1.5 x 10(4) M(-1) for the adenine bases. On the other hand, Ag-RNA adducts showed one binding site with K = 1.5 x 10(5) M(-1) for the guanine bases.     

Interaction of DNA with divalent metal ions

The interaction between the native DNA macromolecules and Ca2+, Mn2+, Cu2+ ions in solutions of low ionic strength (10(-3) M Na+) is studied using the methods of differential UV spectroscopy and CD spectroscopy. It is shown that the transition metal ions Mn2+ exercise binding to the nitrogen bases of DNA at concentrations approximately 5 x 10(-6) M and form chelates with guanine of N7-Me(2+)-O6 type. Only at high concentrations in solution (5 x 10(-3) M) do Ca2+ ions interact with the nitrogen bases of native DNA. In the process of binding to Ca2+ and Mn2+ the DNA conformation experiences some changes under which the secondary structure of the biopolymer is within the B-form family. The DNA transition to the new conformation is revealed by its binding to Cu2+ ions.     

A comparative study of DNA complexation with Mg(II) and Ca(II) in aqueous solution: major and minor grooves bindings

Although structural differences for the Mg-DNA and Ca-DNA complexes are provided in the solid state, such comparative study in aqueous solution has been less investigated. The aim of this study was to examine the bindings of Mg and Ca cations with calf thymus DNA in aqueous solution at physiological pH, using constant concentration of DNA (1.25 or 12.5 mM) and various concentrations of metal ions (2 microM-650 microM). Capillary electrophoresis, UV-visible, and Fourier transform infrared spectroscopic methods were used to determine the cation-binding modes, the binding constants, and DNA structural variations in aqueous solution. Direct Ca-PO(2) binding was evident by major spectral changes (shifting and splitting) of the backbone PO(2) asymmetric stretching at 1222 cm(-1) with K = 4.80 x 10(5) M(-1), whereas an indirect Mg-phosphate interaction occurred (due to the lack of shifting and splitting of the phosphate band at 1222 cm(-1)) with K = 5.6 x 10(4) M(-1). The metal-base bindings were directly for the Mg with K = 3.20 x 10(5) M(-1) and indirectly for the Ca cation with K = 3.0 x 10(4) M(-1). Both major and minor groove bindings were observed with no alteration of the B-DNA conformation.