脱氧核糖核酸在电极表面的固定化研究进展

介绍了电化学脱氧核糖核酸(DNA)传感器的原理并重点评述了DNA在不同材料电极表面的固定化技术,对比了吸附法、自组装法、亲和素一生物素法、共价键合法以及组合法固定DNA在构建电化学DNA传感器中的应用。参考文献77篇。     

单链脱氧核糖核酸探针在氧化锆陶瓷小珠表面的微阵列及陶瓷荧光小珠的制备

以N－（4－马来酰亚胺氧化丁酰）－琥珀酰亚胺磺酸钠盐（sulfo-GMBS)为介剂将5′－氨基／3′－得克萨斯红双末端修饰DNA探针偶联到3－（2－氨乙基氨丙基）三甲氧基硅烷（AATS）修饰的300μmZrO2陶瓷小珠表面，制成荧光小珠，小珠表面上单链DNA探针分子之间的距离取决于小球表面的预处理、偶联以应溶液中DNA探针浓度，小珠大小和小珠的数目。在优化条件下，20-mer单链DNA探针在小珠表面微阵列的最小分子间距约为7．6nm，即在一个300μmZrO2陶瓷小珠表面有10^10数量级的20－mer单链DNA探针参与微阵列反应。机理研究表明：DNA探针通过sulfo-GMBS偶联到小珠表面的化学反应仅涉及到探针末端修饰氨基。     

单链脱氧核糖核酸探针在羧基聚苯乙烯小珠表面的微阵列条件及固—液杂交特

优化在１－乙基－３－（３－二甲基氨丙基）－碳化二亚胺（ＥＤＣ）存在下,５′－ＮＨ２单末端和５′－ＴＲ／３′－ＮＨ２双末端修饰单链脱氧核糖核酸（ＤＮＡ）探针在１０μｍ羧基聚苯乙烯小珠表面进行微阵列的条件,研究５′－ＮＨ２单末端修饰ＤＮＡ探针与其碱基互补充阢进行的固－液杂交特性。结果表明,在ＰＨ４．６－５．６,ＥＤＣ浓度为０．５－０．７ｇ／Ｌ时,ＤＮＡ探针在小珠表面具有最大的微阵列能力。微阵列在小珠表面的５′－ＨＮ２单末端修饰ＤＮＡ探针,与期 碱基互补序列的杂交反应遵守二级反应动力学,杂交优化条件除取决于溶液介质的ＰＨ值,离子强度和碱基互补序列 溶液介质中的浓度外,还取决于环境温度和杂交时间。测定了２５℃下微阵列在小珠表面的２０－ｍｅｒＤＮＡ探针与其溶液中互补序列的杂交反应常数。     

毛细管电泳与MFOLD软件研究单链脱氧核糖核酸分离二级结构

从Gene Bank数据库中下载质粒基因序列,用DNAssist软件分析并最终建立了单点碱基突变模型,并以此为研究对象对单链DNA分离机理进行研究探讨。采用高效毛细管电泳-激光诱导荧光检测(CE-LIF)方法,以线性聚丙烯酸胺为筛分介质对模型进行分离分析,并与MFOLD软件所预测的单链二级结构进行对照分析。分离条件为:线性聚丙烯酸胺浓度为6%,负极进样,13kV分离,温度为19℃,λem=488nm、λex=520nm。结果发现毛细管电泳实际分析结果有4个单链峰,而MFOLD软件预测的只有3种二级结构,预测的准确度为75%,MFOLD软件虽然有局限性,但预测DNA二级结构仍有一定的参考价值。     

铂电极表面生物素-亲和素固载单链脱氧核糖核酸的电化学传感器

通过直接吸附将亲和素固定在Pt电极表面,联于生物素标记的脱氧核糖核酸(DNA)探针,制备了电化学基因传感器,建立了Pt电极表面修饰单链脱氧核糖核酸(ssDNA)的方法。修饰电极与待测溶液中人工合成的转基因食品中常有的花椰菜花叶病毒35S启动子(CaMV35S)或根癌农杆菌终止子(NOS)DNA片段进行杂交,以邻菲罗林钴络合物[Co(phen)3^3 ]为杂交指示剂,循环伏安法测量,通过杂交前后指示剂峰电流的变化检测DNA杂交的量。研究了电极修饰、杂交反应及测量的适宜条件,在优化实验条件下,峰电流的差值与DNA杂交量之间有良好的线性关系,相关系数r=0．9996。杂交后的电极经热变性再生,可重复使用多次。     

三螺旋脱氧核糖核酸的研究进展

阐述了三螺旋DNA的发展和最新动态,并从3个方面展开评述：(1)三螺旋DNA稳定性的研究。三螺旋DNA的稳定性不仅取决于寡聚核苷酸的内在结构,还受外界环境如溶液的pH值、阳离子的种类和价态、DNA分子嵌入试剂和共聚物等的影响;(2)三螺旋DNA的应用。三螺旋DNA的形成为基因操纵和基因疗法提供了新方法,它在抑制DNA转录、复制、基因定点诱变、定点切割和诱导基因重组等方面有重要的应用前景;(3)三螺旋DNA的检测方法,包括紫外-可见吸收光谱法、荧光分析法、原子力显微术和顺磁共振谱等。     

修饰石墨电极表面电化学发光反应微环境的研究及其分析应用

用电化学方法对石墨电极表面进行预阳极化修饰后,该修饰电极不但能选择性地使卡托普利富集于修饰电极表面,而且还创造了一个适合于卡托普利显著增敏钌(Ⅱ)联吡啶体系的电化学发光信号的微环境。在优化的实验条件下,该方法的线性范围为2.0×10-8-8.0×10-6g/L,检出限达5.0×10-9g/L。该方法成功应用于尿样的检测。     

自组装膜中原位光反应固定化脱氧核糖核酸及膜稳定性

通过静电沉积的方法构筑了含有脱氧核糖核酸(DNA)和重氮树脂(DAR)的交替多层膜.在紫外光照射下,这种静电沉积多层膜相邻层间DNA上的磷酸基团与DAR上的重氮基团发生反应,从而将DNA共价连接到多层膜中.利用紫外-可见吸收光谱和掠角反射吸收傅里叶变换红外光谱(GRAFTIR)研究了这种相邻层间的光化学变化.刻蚀实验结果发现,与光照前相比,光照后的膜在盐溶液中的稳定性大大增强.     

基于DNAzyme的单链核酸酶活性研究

本文报道了一种基于DNAzyme的可视检测单链核酸酶活性的新方法.DNAzyme是一种具有类过氧化物酶活性的单链DNA分子,在H_2O_2存在下能够催化无色底物2,2′-连氮基-双-(3-乙基并二氢噻唑啉-6-磺酸)二价阴离子(ABTS~(2-))氧化成蓝绿色物质ABTS~-·5自由基.催化体系中单链核酸酶的加入能水解DNAzyme,导致被DNAzyme催化的ABTS~-·5减少,从而可以通过颜色变化和紫外-可见吸收光谱检测相应的单链核酸酶活性.以Dnase I和S1核酸酶作为单链核酸酶代表进行实验,实验结果表明对Dnase I检测的线性范围为0.5 ～5 U/mL,检出限为0.15 U/mL;对S1核酸酶检测的线性范围为1～10 U/mL,检出限为0.11 U/mL.该方法还能用于单链核酸酶抑制剂的检测,结果表明:Zn~(2+)对Dnase I的半数抑制浓度(Ic_(50))为56.4 mol/L,焦磷酸盐对S1核酸酶的Ic50为1.17 mmol/L.     

石墨电极的纯度对直读光谱分析的影响

石墨电极的纯度对光谱分析的影响只是在一些书中提及,表作试验研究和分析,对电极的纯度的检验未作有效论述。文中就此问题提出了作者的见解,拟定了检验电极纯度的方法。     

脱氧核糖核酸电化学传感器的原理及其应用

对电化学DNA传感器的组成及其在DNA损伤研究、环境污染监控、病原基因检测、基因疾病诊断和药物机理分析等方面的进行了总结，并对其发展趋势进行了评述。     

Electrochemical Determination of Oxalate at Pyrolytic Graphite Electrodes

The electrocatalytic oxidation of oxalate at several carbon based electrodes including basal plane (BPPG) and edge plane (EPPG) pyrolytic graphite and glassy carbon (GC) electrode, was studied. The electrodes were examined for the sensing of oxalate ions in aqueous solutions and all three electrodes showed a response to oxalate additions. The peak of oxalate oxidation at BPPG electrode appeared at lower potential, +1.13 V vs. SCE, than at EPPG (+1.20 V vs. SCE) and GC electrode (+1.44 V vs. SCE). Oxalate oxidation at BPPG electrode was studied in more details for response characteristics (potential and current), effects of pH, temporal characteristics of response potential and current. The results indicated that oxalate oxidation proceeds as two‐electron process at the BPPG electrode with a transfer coefficient β and a diffusion coefficient D evaluated to be 0.45 and 1.03 (±0.04)×10^?5 cm² s^?1 respectively. The BPPG electrode was found to be suitable for oxalate determination in aqueous media showing linear response to oxalate concentration with a sensitivity of 0.039 AM^?1 and a limit of detection of 0.7 μM.     

Direct Oxidation of Ascorbic Acid at an Edge Plane Pyrolytic Graphite Electrode: A Comparison of the Electroanalytical Response with Other Carbon Electrodes

The direct electrochemical oxidation of ascorbic acid at an edge plane pyrolytic graphite electrode (EPPG) is investigated and compared with other common carbon‐based electrodes, specifically glassy carbon, boron doped diamond and basal plane pyrolytic graphite. It is found that the EPPG electrode shows a significantly higher degree of electrochemical reversibility than the other electrode substrates giving rise to an analytically optimized limit of detection and sensitivity of 7.1×10^?5 M and 0.065 A M^?1 respectively.     

Simultaneous Determination of Uric Acid and Ascorbic Acid Using Edge Plane Pyrolytic Graphite Electrodes

Edge plane pyrolytic graphite electrodes have been applied for the determination of uric acid and ascorbic acid. The separate determination of uric acid was found to produce three linear ranges from 100 nM to 3400 μM with a detection limit of 30 nM found to be possible. Uric acid detection was also explored in the presence of 200 μM ascorbic acid where a detection limit of 52 nM was found to be possible. The detection of ascorbic acid in the presence of uric acid was also explored over three linear ranges of ascorbic acid with a limit of detection of 80 nM. Last the simultaneous determination of both uric acid and ascorbic acid is investigated over the range 100 nM to 1000 μM where detection limits of 50 nM and 120 nM were obtained respectively. Analysis of uric acid in a growth tissue medium was found to be successful, confirming the applicability of the methodology to real matrices. This protocol is shown to provide low detection limits, easy handling (no electrode modification), good voltammetric peak separation of uric acid and ascorbic acid and a wide linear dynamic range.     

脱氧核糖核酸电分析化学研究进展

就DNA电分析化学研究及其应用方面进行综述，主要叙述了DNA的各种电分析化学方法，以及DNA电化学传感器的原理，应用以及发展，本文引用文献77篇。     

Immobilization of Heparin and Highly-Sulphated Hyaluronic Acid onto Plasma-Treated Polyethylene

Heparin and highly-sulphated hyaluronic acid have been successfully immobilized onto plasma-processed polyethylene via a diamine polyethyleneglycol (PEG) spacer molecule. Two different plasma-processes have been utilized, i.e. a treatment and a deposition process, for providing polyethylene surface with the COOH groups necessary for the immobilization reactions. XPS integrated with derivatization procedures, ATR-FTIR and Water Contact Angle measurements have been carried out for characterizing each modification step: 1) the plasma-process, 2) the immobilization of the spacer molecule and 3) the immobilization of the biomolecules. The thrombin time of the modified surfaces has been measured, and their platelet activation characteristics evaluated. The results indicate a certain nonthrombogenic character of the biomolecule-immobilized polyethylene samples.     

丁基锡系列化合物与脱氧核糖核酸的相互作用

通过紫外（ultraviolet,UV）光谱和圆二色（circular dichroism,CD）光谱,研究了丁基锡化合物（一丁基锡、二丁基锡和三丁基锡）与脱氧核糖核酸（deoxyribonucleic acid,DNA）的作用方式以及时间和浓度的影响。结果显示丁基锡化合物与DNA的作用是双重的,既作用于DNA的碱基,对双螺旋结构有一定影响,又作用于DNA的磷酸基团,使构象发生变化。但是,丁基锡化合物与脱氧核糖核酸作用的程度和方式与丁基锡种类、时间和浓度等因素有关。一丁基锡倾向于与磷酸基团作用,三丁基锡倾向于与碱基作用,而二丁基锡与两者作用程度相近。短时间内,丁基锡化合物的作用位点通常是DNA的碱基;长时间时,则作用位点往往是DNA的磷酸基团。低浓度的丁基锡化合物倾向与DNA的碱基结合,高浓度的丁基锡化合物倾向与DNA的磷酸基团结合。     

十六烷基溴化吡啶与脱氧核糖核酸作用的共振光散射光谱及其分析应用

在碱性条件下，十六烷基溴化吡啶(CPB)与脱氧核糖核酸(DNA)共存时，体系产生较强的共振光散射，其强度与DNA浓度呈线性关系，据此提出了基于阳离子表面活性剂的共振光散射法定量测定DNA。在最佳实验条件下，测得小牛胸腺DNA(ctDNA)和鱼精子DNA(fsDNA)的线性范围分别为0．2-2．0mg／L和0．2—1．25mg／L，检出限分别为0．07mg／L和0．05mg／L。该方法已应用于合成样品及实际样品中DNA含量的测定。