Determination of Azidothymidine – an Antiproliferative and Virostatic Drug by Square‐Wave Voltammetry

The 3′‐azido‐3′‐deoxythymidine (AZT, Zidovudine) is an antiproliferative and virostatic drug widely used in human immunodeficiency virus type 1 (HIV‐1) infection treatment. With respect to side effects of high doses and a short half‐life of AZT, a fast and simple detection method for this agent could be helpful. The aim of our study was to determine AZT levels in natural samples (urine, serum, whole blood, and cell cultures, such as the HaCaT line of keratinocytes) without their mineralization and/or purification, by means of electrochemical methods using hanging mercury drop electrode (HMDE). On this electrode, AZT undergoes irreversible reduction at the peak potential near E_p?1.1 V (vs. Ag/AgCl/3 M KCl). Reduction AZT signals were measured by cyclic voltammetry (CV), differential pulse voltammetry (DPV), square‐wave voltammetry (SWV), and constant current chronopotentiometric stripping analysis (CPSA). In phosphate buffer (pH 8) the SWV yielded the best AZT signal with the detection limit of 1 nM. The determination of AZT concentration in biological materials is affected by electroactive components, such as proteins and DNA. For monitoring the influence of these compounds, AZT reduction was performed in the presence of 10 μg/mL calf thymus ssDNA and/or 100 μg/mL bovine serum albumin. In these cases, the detection limit increased to 0.25 μM. Also studied was the AZT concentration in keratinocyte cells (HaCaT line) during cell cultivation. It has been shown that the SWV may be considered as a useful tool for the determination of AZT concentration in cell cultures, and for monitoring AZT pharmacokinetics.     

Rapid on-chip postcolumn labeling and high-resolution separations of DNA

When performing genetic analysis on microfluidic systems, labeling the sample DNA for detection is a critical preparation step. Labeling procedures often involve fluorescently tagged primers and PCRs, which lengthen experimental run times and introduce higher levels of complexity, increasing the overall cost per analysis. Alternatively, on-chip labeling techniques based on intercalating dyes permit rapid labeling of DNA fragments. However, as noted in the literature, the stochastic nature of dye-DNA complex formation hinders the native electrophoretic migration of DNA fragments, degrading the separation resolution. In this study, we present a novel method of controllably labeling DNA fragments at the end of the electrophoretic separation channel in a glass microfluidic chip. Permitting the DNA to separate and labeling just before detection, achieves the rapid labeling associated with intercalators while maintaining the high resolution of native DNA separations. Our analyses are completed in minutes, rather than the hours typical of sample prelabeling. We demonstrate an electrophoretic microchip-based intercalator labeling technique that achieves higher resolution performance than reported in the literature to date.     

Isolation, Mapping, DNA Sequence and RFLPs Studies of Random Single-Copy DNA Segments on Human X Chromosome

Using the total human/mouse DNA as the probe, screening has been carried out three times with in situ plaque hybridization to obtain the single-copy DNA sequence from the human X chromosome genomic library. The effective rate of screening is 1. 45%. DNAs from clones containing single-copy inserts have been analyzed by a panel of hybrid cells with or without human X chromosome. Three segments, designated by DXFD52,73,75, are mapped to the X chromosome. DXFD52 has been precisely localized on Xq12-q13 with in situ chromosomal hybridization. DXFD52 has been partially sequenced. The results indicate that DXFD52 is a new isolated single-copy segment on the X chromosome. Great progress in the RFLPs study with DXFD52 has been achieved in the population of Chongqing, Sichuan Province. The results show that the DXFD52 can be used to detect the RFLP with Hind Ⅲ, Bgl Ⅱ, and Hinf Ⅰ. DXFD52 will be a potential "landmark" for the construction of the complete linkage map of human genome and the analysis of genomic s     

Effect of solvent on the cation-sensitive fluorescence of polyanions bearing 4′-acryloylbenzo-18-crown-6 Units

Changes in the fluorescence intensity of polyanions bearing 4-acryloylbenzo-18-crown-6 units on the addition of cations were studied in a mixed solvent of methanol and water at 30°C. The sensitivity of the change in fluorescence intensities of the polymers toward cations was strongly enhanced compared to that of the corresponding model compound. When alkali metal cations were added, the fluorescence intensity of the polymers decreased in the orders Li⁺>Na⁺>Cs⁺>Rb⁺>K⁺ in a methanol-water (19) mixture and Li⁺>Na⁺>Rb⁺>K⁺Cs⁺ in a methanol-water (91) mixture. Alkaline earth metal cations and alkylamine hydrochlorides decreased the fluorescence intensity of the polymers in a methanol-water (19) mixture. The cation-dependent decrease in the fluorescence intensity of the polymers was affected by the water fraction in a mixed solvent of methanol and water.     

SNPs Feasibility of Nonlabeled Oligonucletides on Using Electrochemical Sensing

We have demonstrated an electrochemical gene chip protocol for the SNPs detection of nonlabeled DNA. Using an array consisting of streptavidin‐modified gold electrodes, probe DNA were attached through the application of a direct electric field. Electrochemical response changes originating from the hybridization of nucleic acids to protein‐bound nucleic acids using soluble mediators in K₃Fe(CN)₆ solution could then be observed. The electrochemical protocol developed showed high sensitivity and good reproducibility in the detection of DNA hybridization. Significant changes in electrochemical signals were also observed when using target DNA with a single base mismatch, indicating the applicability of this method to single nucleotide polymorphisms (SNPs) detection.     

Interaction of the mutagen ethidium bromide with DNA, using a carbon paste electrode and a hanging mercury drop electrode

The interaction of ethidium bromide (2,7-diamino-10-ethyl-9-phenylphenanthridinium bromide; EB) with double stranded (ds) calf thymus DNA and thermally denatured single stranded (ss) DNA was studied in solution and at the electrode surface by means of transfer voltammetry using a carbon paste electrode (CPE) as working electrode in 0.2 M acetate buffer, pH 5.0. As a result of intercalation of this dye between the base pairs of dsDNA, the characteristic peak of dsDNA, due to the oxidation of guanine residues, decreased and after a particular concentration of EB a new peak at +0.81 V appeared, probably due to the formation of a complex between dsDNA and EB. The non-intercalated EB gives another peak, but at an increased concentration of the dye. A similar behaviour was observed during the interaction of the dye with ssDNA.Furthermore, the interaction of EB with ds, ss and supercoiled (sc) DNA was studied at the hanging mercury drop electrode (HMDE) surface by means of alternating current voltammetry in 0.3 M NaCl and 50 mM sodium phosphate buffer (pH 8.5) as supporting electrolyte. dsDNA yields a smaller peak at −1.42 V (peak III) compared to the one yielded by ssDNA, since the latter is a relaxed and more accessible form. By addition of EB into the buffer solution an increase of peak III was observed in the dsDNA form as well as in ssDNA resulting from their interaction with EB. Furthermore, the appearance of peak III in covalently closed circular scDNA after exposure to increasing concentrations of EB is a result of the introduction of ‘free ends’ in DNA affecting its structural integrity.     

三菲咯啉合铁手性配合物键合DNA的立体选择性

应用光谱法研究了手性金属配合物［Ｆｅ（ｐｈｅｎ）３］＾２＋与小牛胸腺ＤＮＡ的作用，确定了△型异构体对Ｂ型右手螺旋ＤＮＡ有优先键合的立体选择性。     

MgCl2 Enhances Cluster Formation by Nanoscale Toroidal DNA Condensates

Multivalent cations can cause DNA to condense from its extended state in solution into high-density toroid-shaped particles. Developing methods to control the size and size distribution of DNA toroids is an important goal for the development of artificial gene delivery systems. Here we demonstrate that changes in salt conditions, prior to condensation by multivalent cations, can significantly affect DNA condensation. Specifically, millimolar concentrations of MgCl₂ are shown to cause the formation of toroid clusters, whereas NaCl at the same ionic strength does not.     

红四氮唑作为电化学嵌合剂的核酸杂交生物传感器

提出了一种以红四氮唑 (TTC)作为嵌合剂 ,采用石墨修饰电极监测核酸杂交过程和测定特定 DNA片段的灵敏的电化学方法 . TTC具有背景电流低 ,电活性强和对双链 DNA(ds DNA)选择性好等一系列优点 ,可在石墨电极表面形成 ds DNA-TTC层 .在电化学测定中 ,TTC的还原电流与靶 DNA浓度具有良好的线性关系 ,对 DNA的检测限可低达 6× 1 0 - 1 1 mol/L .