DNA-纳米多孔羟基磷灰石修饰电极用于研究DNA与亚甲基蓝的相互作用

利用溶胶-凝胶法将具有优良生物相容性和独特吸附性的羟基磷灰石(HAp)修饰在玻碳电极上形成纳米多孔薄膜. 电化学实验结果证明该纳米多孔羟基磷灰石薄膜能有效地将双链DNA吸附于其表面. 采用循环伏安法系统研究了固定在HAp薄膜上的DNA与亚甲基蓝(MB)之间的相互作用. 实验结果表明, 在20～200 mV•s^－1扫描速度范围内该电极反应过程系表面反应控制; 在pH 6.0～7.4范围内, MB在DNA修饰电极上的峰电位随pH的增加而向负方向移动; 当磷酸盐缓冲溶液中的离子强度小于59 mmol•L^－1时, MB与DNA之间为静电作用, 当离子强度大于59 mmol•L^－1时, 二者之间既有静电作用, 也有部分嵌入作用. 根据Langmuir吸附公式, 得出MB与DNA之间的结合常数为4.2×10⁴ mol^－1•L.     

DNA／聚（3-甲基噻吩）复合膜修饰电极的制备及其应用于研究8-羟基-2＇-脱氧鸟嘌呤核苷的伏安行为及测定

首先通过电聚合方法在玻碳电极表面制备了聚（3-甲基噻吩）（P3MT）修饰膜,然后在一定电位下将DNA分子电沉积到P3MT表面,制备了DNA／（P3MT）复合膜修饰玻碳电极．研究了8-羟基-2’-脱氧鸟嘌呤核苷（8-OH-dG）在该复合膜修饰电极上的伏安行为以及扫描速度、pH值和尿酸对其伏安行为和检测的影响．实验结果表明,该复合膜修饰电极结合了P3MT和DNA两者的优点,使8-OH-dG在复合膜修饰电极上的电化学行为明显改善,而且具有很好的重现性和稳定性．在0．1mol／LpH7．0的磷酸盐缓冲溶液（PBS）中,8-OH-dG的氧化峰电流与其浓度在0．28～4．2μmol／L和4．2～19．6μmol／L两个范围内成良好的线性关系,检出限为56nmol／L（S／N=3）．该研究可以为制备HPLC或毛细管电泳电化学检测器检测8-OH—dG打下一定的基础,因此在检测尿样中8-OH-dG的研究方面具有潜在的应用价值．     

毛细管电泳-修饰电极电化学检测法测定饮用水中溴酸盐

通过静电组装技术在碳圆盘电极（PGE）表面制备{聚二烯丙基二甲基氯化铵（PDDA）/多壁碳纳米管（MWCNT）}n/PDDA多膜,并采用循环伏安法在多膜表面电化学修饰一磷钼酸（PMo12）膜,构筑PGE/{PDDA/MWNTs}5/PDDA/PMo12复合膜修饰电极,研究该复合膜修饰电极电化学及其对溴酸盐（BrO3-）电催化还原性质.在此基础上建立毛细管电泳-PGE/{PDDA/MWNTs}5/PDDA/PMo12修饰电极电化学检法定饮用水中溴酸盐分析新方法.在优化实验条件下,电泳峰面积与溴酸根浓度在5.0×10-8～5.0×10-5mol/L范围内呈良好性关系（r=0.9954）,检出为2.0×10-8mol/L（S/N=3）.     

灿烂甲酚蓝在DNA修饰金电极上的电化学行为

利用自组装技术将巯基乙醇固定在金电极表面形成巯基乙醇自组装膜修饰金电极, 用乙基-(3-二甲基氨丙基)碳二亚胺盐酸盐(EDC)和N-羟基琥珀酰亚胺(NHS)为偶联试剂, 分别将鲱鱼精单链DNA(ssDNA)和双链DNA(dsDNA)固定于金电极表面形成ssDNA和dsDNA 修饰电极. 考察了灿烂甲酚蓝(BCB)在不同DNA 修饰电极上的电化学行为,结果表明, BCB 在ssDNA 和dsDNA 修饰电极上的吸附常数分别为1.67×10^4和3.22×10^4 L·mol-1, BCB 与ssDNA 主要以静电作用结合, 而与dsDNA作用存在静电和嵌插两种模式. dsDNA 对BCB 具有更高的亲和力, 使BCB 可以作为一种有效的电化学杂交指示剂.     

石墨烯修饰玻碳电极用于急性早幼粒细胞白血病PML/RARa融合基因的检测

应利用电化学还原法将固定在玻碳电极表面的氧化石墨还原为石墨烯,然后再利用偶联活化剂将氨基修饰的急性早幼粒细胞白血病（APL）PML／RARα融合基因序列探针固定到石墨烯修饰电极表面,以亚甲基蓝（MB）为电化学杂交指示剂,并由差分脉冲伏安法检测人工合成APL的PML／RARα融合基因.结果表明,石墨烯对MB的检测信号起到了很好的增敏作用,杂交前后MB还原峰电流差值与靶标链DNA浓度在5×10-10~2.5×10-9 mol/L范围内呈线性关系,检出限为8×10-11 mol/L.该方法简单、特异性好,有望用于实际样品的检测.     

基于单壁碳纳米管-十二醛复合材料的DNA电化学传感器

将单壁碳纳米管(SWNTs)和十二醛(DA)混合超声分散,得到均匀、稳定的无机-有机纳米复合材料(SWNTs-DA)。将其滴涂在玻碳电极表面晾干得到复合材料修饰电极(SWNTs-DA/GCE),再通过胺醛缩合反应将末端修饰氨基的单链DNA探针共价固定在SWNTs-DA/GCE表面,构建了一种新型的DNA电化学传感器。以[Fe(CN)6]3-/4-为电活性探针,采用循环伏安法和电化学阻抗法对传感器的层层组装过程进行表征。以亚甲基蓝(MB)作为杂交指示剂,考察了传感器分析性能。实验结果表明,MB在传感器上的峰电流值(Ip)与互补序列浓度对数值(lgcS2)在1.0×10-15～1.0×10-10mol/L范围内呈良好的线性关系(r=0.998)。根据3倍信噪比(S/N=3),计算得检出限为2.0×10-16mol/L。选择性实验表明该传感器能对互补序列、三碱基错配序列和非互补序列进行很好的识别。     

几种表面活性剂与DNA的相互作用

用循环伏安、紫外－可见光谱和交流阻抗等方法，以电活性小分子亚甲基蓝（ MB）为探针，研究了几种表面活性剂与DNA的相互作用。研究发现，阴离子、阳离 子和非离子表面活性剂均可通过疏水和静电作用与固定在电极表面的DNA分子结合 ，改变电极表面DNA的状态，进而影响电活性小分子的电化学行为。阴离子表面活 性剂与DNA之间以静电排斥为主，也有部分疏水性结合，它使MB的氧化还原峰峰电 流减小。阳离子表面活性剂十六烷基三甲基溴化铵、十二烷基三甲基氯化铵均在一 定浓度范围内对MB的电化学响应有增敏作用，而代十六烷基吡啶、溴代十八烷基吡 啶表现出抑制效应，它们与DNA间既有疏水性作用，也有静电吸引。非离子表面活 性剂与DNA的结合较弱，其主要是通过改变溶液的性质（如粘度、极性和介电常数 等）影响DNA的构象，从而导致MB电化学参数的微弱变化。此外，表面活性剂疏水 链的长短及极性头基的大小对作用过程也有一定影响。     

聚苯胺纳米线修饰的DNA电化学传感器的研究

采用电流密度递减的方法在玻碳电极表面修饰聚苯胺纳米线(PAINW), 以SEM对其形貌进行表征, 测得PAINW的尖端直径在80～100 nm之间. 以乙基-(3-二甲基丙基)碳化二亚胺盐酸盐(EDC)为偶联活化剂, 将5'-磷酸基修饰的寡聚核苷酸片断共价固定在聚苯胺修饰的电极上, 一定条件下, 以亚甲基蓝为电化学杂交指示剂, 采用差分脉冲伏安法对杂交信号进行检测, 实现了对特定序列DNA片段的互补、非互补序列的识别.     

电化学方法研究芦丁与鲱鱼精DNA之间的相互作用

采用循环伏安法和交流阻抗法研究了固定在羟基磷灰石薄膜上的DNA与芦丁之间的相互作用.在pH 5.6～7.0范围内,芦丁在DNA修饰电极上的峰电位随pH的增加向负方向移动;在50～800 mV/s扫描速度范围内电极过程同时受扩散和吸附控制,且扩散控制占主导作用;随溶液离子强度增大,芦丁在DNA修饰电极上的表观式量电位不断正移,表明芦丁与DNA之间存在一定的嵌入作用,二者结合形成了超分子化合物.     

基于室温离子液体的有序多孔金膜葡萄糖传感器

将1-丁基-3-甲基咪唑四氟硼酸盐（[BMIm][BF4]）、N,N-二甲基甲酰胺（DMF）与葡萄糖氧化酶（GOD）的混合物修饰于三维有序大孔（3DOM）金膜电极上,构建了一种新型的葡萄糖传感器．固定的GOD在pH7．0的磷酸缓冲液（PBS）中展现出一对可逆性好的氧化还原峰,这归因于GOD的活性中心黄素腺嘌呤二核苷酸（FAD）的直接电化学行为．研究表明,离子液体（IL）、DMF以及3DOM金膜对GOD的直接电化学都起到了重要的作用．3DOM金膜修饰电极作为基底提高了酶的负载量,加速了GOD与电极表面的电子传递;IL的应用增加了固定GOD的电化学活性;DMF与IL、GOD的协同作用更好地保持了GOD的生物活性．固定在电极表面的GOD对葡萄糖显示出良好的催化性能,其检测线性范围为10～125nmol／L,检测限为3．3nmol／L（S／N=3）,酶催化反应的表观米氏常数Km为0．018mmol／L．     

Ligating DNA with DNA

Cloning DNA typically involves the joining of target DNAs with vector constructs by enzymatic ligation. A commonly used enzyme for this reaction is bacteriophage T4 DNA ligase, which requires ATP as the energy source to catalyze the otherwise unfavorable formation of a phosphodiester bond. Using in vitro selection, we have isolated a DNA sequence that catalyzes the ligation of DNA in the absence of protein enzymes. We have used the action of two catalytic DNAs, an ATP-dependent self-adenylating deoxyribozyme (AppDNA) and a self-ligating deoxyribozyme, to create a ligation system that covalently joins oligonucleotides via the formation of a 3',5'-phosphodiester linkage. The two-step process is conducted in separate reaction vessels wherein the products of deoxyribozyme adenylation are purified before their use as substrates for deoxyribozyme ligation. The final ligation step of the deoxyribozyme-catalyzed sequence of reactions mimics the final step of the T4 DNA ligase reaction. The initial rate constant (k(obs)) of the optimized deoxyribozyme ligase was found to be 1 x 10(-)(4) min(-)(1). Under these conditions, the ligase deoxyribozyme promotes DNA ligation at least 10(5)-fold faster than that generated by a simple DNA template. The self-ligating deoxyribozyme has also been reconfigured to generate a trans-acting construct that joins separate DNA oligonucleotides of defined sequence. However, the sequence requirements of the AppDNA and that of the 3' terminus of the deoxyribozyme ligase limit the range of sequences that can be ligated.     

DNA polymerase-catalyzed DNA network growth

The distinct base pairing property of DNA is an advantageous phenomenon that has been exploited in the usage of DNA as scaffold for directed self-organization to form nanometer-sized objects in a desirable fashion. Herein we report the construction of three-dimensional DNA-based networks that can be generated and amplified by the DNA polymerase chain reaction (PCR). The approach is flexible allowing tuning of the meshes of the network by variation of the size of the template. Additionally, further diversification can be introduced by employment of chemically modified nucleotides in PCR allowing the introduction of functionalities and reporter moieties.     

DNA loops and semicatenated DNA junctions

Background  

Alternative DNA conformations are of particular interest as potential signals to mark important sites on the genome. The structural variability of CA microsatellites is particularly pronounced; these are repetitive poly(CA) · poly(TG) DNA sequences spread in all eukaryotic genomes as tracts of up to 60 base pairs long. Many in vitro studies have shown that the structure of poly(CA) · poly(TG) can vary markedly from the classical right handed DNA double helix and adopt diverse alternative conformations. Here we have studied the mechanism of formation and the structure of an alternative DNA structure, named Form X, which was observed previously by polyacrylamide gel electrophoresis of DNA fragments containing a tract of the CA microsatellite poly(CA) · poly(TG) but had not yet been characterized.     

Electrontransfer through DNA and metal-containing DNA

DNA is currently explored as a new material for functional, molecular nano-architectures. In this respect, one major question is to transform DNA into a conducting material which has the potential for self-assembly into electronically active networks. The article covers recent insight into how DNA transports positive (holes) and negative (excess electrons) charges. It was found that holes move through DNA over significant distances using a G- and to a lesser extent also A-based hopping mechanism. EPR studies and recent investigations with model systems show that excess electrons can also hop through the duplex. The second part of the article describes how DNA is currently modified, particularly coated with metals, in order to increase the conductivity.     

Manipulating DNA writhe through varying DNA sequences

It is demonstrated that the shapes and magnitudes of DNA writhe can be precisely manipulated solely through maneuvering the nucleotide sequence of DNA and without the assistance of topoisomerases.     

New DNA Technology and the DNA Biosensor

Abstract

Recently there has been an increasing demand to produce systems for the detection of specific DNA sequences that are amenable to non-specialised laboratories. This demand has led to many innovative and novel approaches to DNA analysis which may collectively be termed ′new DNA technology′. Here, we review these advances in relation to their applicability for the production of a DNA biosensor. The present state of the art is described and future possibilities are considered.     

QUANTITATION OF DNA DAMAGE IN NON-RADIOACTIVE DNA

Three principal methods have been developed for measuring femtomoles of damage in nanogram quantities of non-radioactive DNA. Lesions which can be quantified include single and double strand breaks, alkali labile sites including apurinic and apyrimidinic sites, and pyrimidine dimers. The first in vitro method measures the conversion of supercoiled DNA to relaxed or linear molecules, and can detect up to four lesions per molecule. The second in vitro method (supercoil depletion) assesses the fraction of intact linear molecules of homogeneous length, and allows detection of 8 lesions/molecule. The third method, measurement of molecular length distributions of DNAs of heterogeneous length, reveals the extent of DNA damage and repair in vivo or in vitro.     

Electrochemical DNA Hybridization Detection Using DNA Cleavage

We report the new method for detection of DNA hybridization using enzymatic cleavage. The strategy is based on that S1 nuclease is able to specifically cleave only single strand DNA, but not double strand DNA. The capture probe DNA, thiolated single strand DNA labeled with electroactive ferrocene group, was immobilized on a gold electrode. After hybridization of target DNA of complementary and noncomplementary sequences, nonhybridized single strand DNA was cleaved using S1 nuclease. The difference of enzymatic cleavage on the modified gold electrode was characterized by cyclic voltammetry and differential pulse voltammetry. We successfully applied this method to the sequence‐selective discrimination between perfectly matched and mismatched target DNA including a single‐base mismatched target DNA. Our method does not require either hybridization indicators or other exogenous signaling molecules which most of the electrochemical hybridization detection systems require.     

DNA origami: the art of folding DNA

The advent of DNA origami technology greatly simplified the design and construction of nanometer-sized DNA objects. The self-assembly of a DNA-origami structure is a straightforward process in which a long single-stranded scaffold (often from the phage M13mp18) is folded into basically any desired shape with the help of a multitude of short helper strands. This approach enables the ready generation of objects with an addressable surface area of a few thousand nm(2) and with a single "pixel" resolution of about 6 nm. The process is rapid, puts low demands on experimental conditions, and delivers target products in high yields. These features make DNA origami the method of choice in structural DNA nanotechnology when two- and three-dimensional objects are desired. This Minireview summarizes recent advances in the design of DNA origami nanostructures, which open the door to numerous exciting applications.