Synchronous fluorescence determination of DNA based on the interaction between methylene blue and DNA

The fluorescence intensity of methylene blue (MB) quenched by DNA in the pH range of 6.5-8.0 was studied with synchronous fluorescence technology. A novel method for detecting single-stranded and double-stranded DNA was developed. The decreased fluorescence intensity at 664 nm is in proportion to the concentration of DNA in the range of 0.28-11.0 μmol L⁻¹ for ctDNA, 0.14-8.25 μmol L⁻¹ for thermally denatured ctDNA and 0.28-8.25 μmol L⁻¹ for hsDNA. The detection limits (S/N = 3) are 0.11, 0.04 and 0.04 μmol L⁻¹, respectively. The method is rapid, selective, and the reagents are lower toxic. It has been used for the determination of DNA in synthetic samples with good satisfaction. In addition, the interaction modes between MB and ctDNA and the mechanism of the fluorescence quenching were also discussed in detail. The experimental results from absorption spectra and fluorescence polarization indicate that the possible interaction modes between MB and DNA are the electrostatic binding and the intercalation binding.     

甲巯咪唑的电化学行为及其与DNA相互作用的研究

建立了多壁碳纳米管修饰玻碳电极(MWNTs/GCE)测定甲巯咪唑(TMZ)的电化学分析新方法,研究了TMZ与DNA的相互作用,探讨了TMZ与DNA相互作用的机理。实验发现TMZ在pH=7.0的磷酸盐缓冲溶液中于0.29V处有一氧化峰,其峰电流与TMZ的浓度在3.0~100μmol/L范围内呈良好的线性关系,检出限(S/N=3)为1.0μmol/L。对30.0μmol/L的TMZ进行11次平行测定,其相对标准偏差(RSD)为3.3%。当不同浓度的鲱鱼精DNA加入TMZ溶液后,其氧化峰电流降低,表明两者发生了插入作用,形成了非电活性化合物。紫外光谱红移增色效应说明TMZ嵌插入DNA双链之间,二者结合比为1∶2,结合常数为9.93×106 L/mol。     

Electrochemical oxidation of morin and interaction with DNA

A poly (tetrafluroethylene)-deoxyribonucleate acid (PTFE-DNA) film-modified glassy carbon electrode (GCE) has been fabricated. The electrochemical oxidation behaviors of morin as well as its interaction with DNA have been studied at PTFE-DNA film-modified GCE and bare GCE by electrochemical methods. This modified electrode shows an enhanced effectiveness towards the oxidation of morin. Importantly, as to the interaction between morin and DNA in solution, characteristic parameters such as the binding stoichiometry and association equilibrium constant according to the Hill model for cooperative binding have been determined on the basis of linear sweep voltammetry and chronocoulometry.     

Electrochemical Studies of the Interaction of 2‐Nitroacridone with DNA and Determination of DNA

A new acridone derivate 2‐nitroacridone (NAD) was synthesized and a new method of electrochemical probe has been proposed for the determination of salmon sperm DNA based on its interaction with NAD. The electrochemical behavior of interaction of NAD with DNA was investigated on glassy carbon electrode (GCE). In the presence of DNA, the peak current of NAD decreases and the peak potential shifts to a more positive potential without appearance of a new peak. The binding ratio between NAD and salmon sperm DNA was calculated to be 2 : 1 and the binding constant was 3.19×10⁵ L/mol. The decrease of the peak current (ΔI_p) of NAD was proportional to the concentration of DNA in the range from 1.55×10^?7 M to 2.02×10^?6 M with the detection limit of 3.10×10^?8 M, and DNA of synthetic sample was determined satisfactorily. Additionally, the binding mechanism was preliminarily discussed. The mode of interaction between NAD and DNA was found to be intercalation binding.     

Chemiluminescence determination of ultramicro DNA with a flow-injection method

A high sensitive flow-injection chemiluminescence method for determination of calf thymus DNA and herring sperm DNA has been developed. The method is based on the chemiluminescence reaction of Rhodamine B-cerium(IV)-thermally denatured DNAs in sulfuric acid media. The proposed procedure allows quantitation of DNAs in the range 2.6×10⁻⁵ to 0.26 μg ml⁻¹ for calf thymus DNA and 5.0×10⁻⁸ to 5.0×10⁻⁵ μg ml⁻¹ for herring sperm DNA with correlation coefficients 0.9998 and 0.9996 (both n=11), respectively. The detection limits (3σ) are 6.5×10⁻⁶ μg ml⁻¹ for calf thymus DNA and 4.3×10⁻⁸ μg ml⁻¹ for herring sperm DNA. The possible mechanism of chemiluminescence in the system is discussed.     

Electrochemical and microscopic studies of surface-confined DNA

We have studied the micropatterning and characterization of the organic monolayers using cyclic voltammetry (CV), scanning electrochemical microscopy (SECM), atom force microscopy, and AC impedance, and have determined the electrochemical parameters, i.e., the apparent reaction rate constant (K _f) and the coverage of the electrode surface (θ). CV and SECM experiments demonstrated that the surface of the modified electrode represents an insulating substrate for ferricyanide. Using the high sensitivity of the electron transfer of ferricyanide to the modification of the gold surface with DNA, we selected this reaction as a probe to study the different modification stages at this modified electrode. SECM images obtained from bare, partially modified, and totally modified electrodes showed very good resolution with different topographies or null according to the extent of modification. Based on a comparison with the results of the experiments, a reasonable agreement can be obtained, which means a conjunction of these techniques.     

Spectroscopic and Electrochemical Studies of the Interaction Between Fuchsin Basic and DNA

Introduction DNAbiosensorsareacompletelynewtypeoftech nologicalconceptionsbyusingspecificaffinitybetween mattersinlivingbeingstodistinguishdirectlyand quicklysequence specificDNA[1].Withtherapidde velopmentofgeneticengineering,oneofthekeyissues needtobere…     

硫堇与DNA分子相互作用的电化学方法研究

采用交流阻抗技术和循环伏安法 ,研究了在硫堇自组装膜修饰金电极上 ,以及在硫堇或DNA吸附修饰的玻碳电极上 ,硫堇与DNA分子的相互作用;硫堇自组装膜修饰金电极与DNA分子作用后 ,阻抗增大 ,表明它们之间发生了作用 ;吸附在玻碳电极上的硫堇 (DNA)与DNA(硫堇 )作用后 ,峰电位和峰电流均发生了变化 ,结合光谱测定结果 ,表明硫堇与DNA分子间存在着嵌插、静电等作用 ,二者作用的反应速度与分子在电极上固定的位置有关;在PBS缓冲液中硫堇 -DNA的表观结合常数为4.9×104L·mol -1 ;交流阻抗技术和循环伏安法是研究小分子与DNA分子间相互作用的经济、快速、简便的方法     

Spectroscopic Electrochemical Studies of Interaction Between Fuchsin Basic DNA

Visible spectroscopic and electrochemical methods were used to study the interactions between DNA and fuchsin basic(FB). FB has an irreversible electro-oxidation peak in 5 mmol/L Tris-HCl buffer solution at pH = 7.4 on a glassy carbon electrode(GCE). After adding certain concentration of dsDNA, the oxidation peak current of FB decreases, but the peak potential hardly changs. The visible absorption spectroscopic study shows that the binding mode of FB to dsDNA is intercalative binding and electrostatic binding when the ratio of the concentration of dsDNA to FB is smaller than 0. 2, and a new substance, which produces a new absorption peak, is obtained via a covalent binding between dsDNA and FB apart from intercalative binding and electrostatic binding when the ratio of the concentration of dsDNA to FB is larger than 0. 2. The visible absorption spectra varies no longer when the ratio of the concentration of dsDNA to FB is larger than 1.5. A mean binding ratio of dsDNA to FB was determined to be 1.4: 1,suggesting that two complexes FB-dsDNA and FB-2dsDNA be formed. The interaction between FB and ssDNA was only electrostatic binding. The more powerful interaction of FB with dsDNA than with ssDNA may be applied for the recognition of dsDNA and ssDNA, and in DNA biosensor as hybridization indicator.     

Electrochemical enzyme-linked immunoassay in a DNA hybridization sensor

In most of the currently developed electrochemical DNA hybridization sensors short single-stranded probe DNA is immobilized on an electrode and both the hybridization and detection steps are carried out on the electrode surface. Here we use a new technology in which DNA hybridization is performed on commercially available magnetic beads and detection on solid electrodes. Paramagnetic Dynabeads Oligo(dT)₂₅ (DBT) with covalently bound (dT)₂₅ probe are used for the hybridization with target DNA containing adenine stretches. Target DNA is modified with osmium tetroxide,2,2′-bipyridine (Os,bipy) and the immunogenic DNA-Os,bipy adduct is determined by the enzyme-linked immunoassay with electrochemical detection. Electroinactive 1-naphthyl phosphate is used as a substrate and the electroactive product (1-naphthol) is measured on the carbon electrodes. Alternatively Os,bipy-modified target DNA can be determined directly by measuring the osmium signal on the pyrolytic graphite electrode (PGE). A comparison between determinations of the 67-mer oligodeoxynucleotide on carbon electrodes using (a) the guanine oxidation signal, (b) direct determination of the DNA-Os,bipy adduct and (c) its electrochemical immunoassay showed immunoassay to be the most sensitive method. In combination with DBT, the DNA hybridization of long target deoxyoligonucleotides (such as 67- and 97-mers) and a DNA PCR product (226-base pairs) have been detected by immunoassay at high sensitivity and specificity.     

一种新型发光探针与DNA相互作用的研究

本文以稳态荧光光谱、紫外-可见吸收光谱、荧光偏振、热变性、阴离子猝灭等手段,研究了一种具有强电荷转移能力的化合物2,7-二[(N-乙基咔唑-3-基)丙烯-1-酮基]芴与DNA的相互作用。研究结果表明,2,7-二[(N-乙基咔唑-3-基)丙烯-1-酮基)芴与DNA的作用方式是混合模式,以嵌插作用为主,同时存在沟槽相互作用,其咔唑基团可能插入到DNA的碱基对之间,结合常数K为8 123.48mol/L,结合位点n为0.71。该发光探针灵敏度高,结合稳定。     

抗坏血酸与DNA相互作用的电化学研究

脱氧核糖酸(DNA)贮存着生物体生命活动的所有信息,这些信息指导着细胞生长、分化、代谢、遗传和变异等重要的生命活动。DNA结构与功能的改变可引起细胞生物学行为的显著改变。DNA与抗癌药物相互作用的研究的报道较多[1,2],而与维生素类药物研究报道相对较少[3]。抗坏血酸(AA)即     

Electrochemical Behaviors of Resveratrol and Its Interaction with DNA

Electrochemical behavior of resveratrol was studied in Britton‐Robinson (B‐R) buffer solution (pH = 4.0) at glassy carbon electrode (GCE) using cyclic voltammetry (CV). Resveratrol showed an irreversible anodic peak at 0.570 V which was involving one electron and one proton. Also, the interaction of resveratrol with double‐stranded fish sperm DNA was investigated by linear sweep voltammetry (LSV) and UV‐vis spectra. The results showed that peak potentials shifted to more positive value and peak currents decreased in electrochemical experiment and the maximum absorption decreased with red shift in UV‐vis spectra experiment with the addition of DNA, indicating the resveratrol interacted with DNA by intercalating into the double helix of DNA. Besides, the binding of resveratrol with DNA, analyzed in terms of the cooperative Hill model, yields the association constant K_a = 3.18 × 10⁵ and a Hill coefficient m = 1.06.     

用紫外光谱和荧光光谱研究三丁基锡化合物与脱氧核糖核酸的相互作用

用紫外光谱和荧光光谱研究了三丁基锡(TBT)化合物与脱氧核糖核酸(DNA)的相互作用。结果表明,在不同条件下,TBT既作用于DNA的磷酸基团,使DNA构象发生变化;又作用于DNA的碱基基团,对DNA双螺旋结构有一定影响。而且,TBT与DNA作用时间越长,则减色越明显。还考察了TBT和磷酸根浓度,以及溶液pH值对TBT与DNA作用的影响。     

Electrochemical and Spectroscopic Study of the Interaction of Indirubin with DNA

The interaction of indirubin with DNA was studied by differential pulse voltammetry (DPV) and linear sweep voltammetry (LSV) at the bare or DNA‐modified electrode and UV‐vis or IR spectra. As a result of intercalating of this drug into the double helical structure of DNA, the DPV of indirubin shows that peak potentials shift and peak currents decrease with the addition of DNA. UV‐vis spectra exhibits that the absorption intensity of indirubin at 538.7 nm^?1 decreases, which indicates that the anticancer herbal drug indirubin binds DNA. In addition, IR‐spectra of DNA and DNA‐indirubin adduct imply indirubin interacts with the phosphate groups of DNA by hydrogen bond or electrostatic interaction. Under our experiment conditions, the decrease of peak current is proportional to DNA concentration, which can be applied to estimate DNA concentration. The results indicate that the herbal drug indirubin can interfere with the DNA by intercalating into the double helix of DNA and interacting with the phosphate groups of DNA.     

Electrochemical studies of NiTMpyP and interaction with DNA

In this paper, cyclic voltammetry, linear sweep voltammetry and chronocoulometry in connection with the hang mercury drop electrode were used to study NiTMpyP and its mixture with DNA. The reduction of NiTMpyP in our experimental conditions involves in 4e reduction of TMpyP. NiTMpyP interacting with DNA forms electrochemically non-active complex DNA-2NiTMpyP, which can not be reduced on the Hg electrode. The peak potential of NiTMpyP does not shift and its electrochemical kinetic parameters indicate no significant change in the presence of DNA. However, the reduction current of NiTMpyP decreases obviously due to the formation of DNA-2NiTMpyP, which implies its equilibrium concentration decreases when DNA was mixed. The decrease of peak current is proportional to DNA concentration, which can be applied to estimate DNA concentration.     

烟酰胺与DNA相互作用的电化学研究

利用示差脉冲伏安法研究了烟酰胺(NA)与小牛胸腺DNA在pH 8.0条件下相互作用的电化学行为.双链DNA(dsDNA)或单链DNA(ssDNA)的存在导致NA的峰电流明显降低且峰电位负移,表明NA与DNA发生相互作用,生成了复合物,且其作用模式主要是静电模式,但NA与dsDNA的相互作用强于与ssDNA的相互作用,可用于识别dsDNA和ssDNA.通过dsDNA加入前后峰电流的变化,计算得出NA与dsDNA结合常数β=4.946×10(11),结合位点数m=3.此外,NA的峰电流Ip与DNA质量浓度在1～14mg/L的范围内呈线性关系,线性回归方程为Ip(10-5A)=-0.03451cDNA(mg/L)+1.7408,相关系数R为0.9998.该法具有良好的回收率和选择性,可用于样品中DNA的测定.     

Electrochemical and Spectroscopic Studies on the Interaction of Gallocyanine with DNA

The interaction of gallocyanine (GC) with double‐stranded DNA (dsDNA) in pH 3.5 Tris‐HCl buffer solution was investigated by electrochemical methods and spectrophotometric methods as well. In the potential scan range of ‐0.25 ? +0.18 V(vs. SCE), GC had a couple of well‐defined redox peaks at ‐0.022 V and ‐0.069 V on a cyclic voltammogram at the scan rate of 100.0 mV/s, respectively. After the addition of dsDNA into the GC solution, the redox‐peak currents decreased obviously and the peak potentials shifted positively. The results demonstrated that GC binding to DNA was caused by intercalation. Electrochemical parameters such as the electron number (n), the charge transfer coefficient (α) and the electrochemical reaction standard rate constant (k_s) were calculated and compared in the absence and presence of dsDNA. Almost unchanged values of the electrochemical parameters after adding dsDNA showed that non‐electroactive complexes were formed when GC interacted with DNA. The results indicated that the decrease of the redox‐peak currents was caused by the decrease of the free concentration of GC in the reaction solution. The binding constant and binding ratio were investigated by spectrophotometric methods. DNA concentration can be determined by the decrease of the peak current of GC. The linear range for dsDNA was in the range of 1.45 × 10^?7 ? 1.45 × 10^?6mol/Land 1.45 × 10^?6 ? 1.45 × 10^?5 mol/L, respectively with the linear regression equation as Δi_P (10^?7 A) = 0.037 + 0.018C (10^?7mol/L), and Δi_P (10^?7 A) = 0.25 + 0.041C (10^?6mol/L), respectively, and the detection limit (3σ) was 1.13 × 10^?7 mol/L.     

Spectral studies on the interaction of Amido black 10B with DNA in the presence of cetyltrimethylammonium bromide

The interaction of Amido black 10B (AB) with DNA in basic medium was studied in the presence of cetyltrimethylammonium bromide (CTMAB) based on the measurements of resonance light scattering (RLS), UV–vis, CD spectra, and RLS imaging. The interaction has been proved to give a ternary complex of CTMAB–DNA–AB in Britton–Robinson buffer of pH 11.55, which exhibits strong negative Cotton effect at 233.3 nm and 642.8 nm, and strong RLS signals characterized at 469 nm. Experiments showed that the enhanced RLS intensities (ΔI_RLS) against the mixture of AB and CTMAB are proportional to the concentration of fish sperm DNA (fsDNA) and calf thymus DNA (ctDNA), respectively over the range of 0.03–1.0 and 0.05–1.5 μg ml⁻¹, with the limits of determination (3σ) of 7.3 ng ml⁻¹ for fsDNA and 7.0 ng ml⁻¹ for ctDNA.