DNA电化学传感器在DNA损伤研究中的应用

利用单链ＤＮＡ分子共价固定在电墨电极表面，采用核酸分子杂交技术，以道诺霉毒作为杂交指示剂形成ＤＮＡ电化学传感器，研究了在不同致突变剂的作用下，特定碱基序列的ＤＮＡ在电极表面能否杂交杂交程度的差异，探讨了ＤＮＡ突变情况及可能的突变机理。     

在石墨电极上脱氧核糖核酸与米托蒽醌嵌入作用的电化学研究

本文研究了单链ＤＮＡ分子在石墨电上的固定方法，采用核酸分子杂交技术，使具有电化学活性的米托蒽酯嵌入ＤＮＡ分子双螺旋结构的碱基对中，在电极上形成ｄｓＤＮＡ－ＭＳ层，通过伏安法研究ＤＮＡ分子和ＭＸ相互作用的电化学行为。     

基于核酸分子杂交的免疫芯片抗体固定新方法

基于核酸分子杂交原理构建了一种新型抗体固定方法.先将抗体与寡核苷酸单链交联,再将两者的复合物与固相载体表面上的互补寡核苷酸链结合,从而将抗体固定到载体的表面.在磁珠表面对该固定方法进行实验,证明了方法的可行性.以本方法构建了针对转基因Bt Cry1Ac蛋白的免疫芯片,用Cy3标记二抗对其探针固定效果进行分析,并且在芯片上对Bt Cry1 Ac蛋白进行梯度浓度检测试验.结果表明,以本方法构建的抗体芯片,探针分布具有良好的特异性;探针层分布均匀,非特异吸附小;检测灵敏度达到0.01 ～ 0.05 μg/L;此外,通过杂交核酸双链的解离成功实现了芯片的再生,有助于解决传统抗体固定方法中芯片不可再生的问题.     

玻璃微流控通道中水凝胶固定寡核苷酸探针的方法及应用

核酸杂交是分子生物学研究中最常用和最基本的分析方法之一．杂交技术有多种,主要区别在于探针的固定．目前常用的是将探针直接固定在载体表面（尼龙膜或硅烷化的玻片）或用磁珠法和水凝胶法固定,其中水凝胶法兼有三维立体和简单实用的优势,其发展颇为引人注意．微流控芯片技术具有集成化和自动化的优势．将水凝胶和微流控技术相结合,将使核酸分析中的杂交、变性以及重新杂交等操作更为简单、快速、易行．     

单链脱氧核糖核酸在石墨电极表面固定化的研究

用５％（Ｖ／Ｖ）３－氨基丙基三乙氧基硅烷（ＰｒＮＨ２硅烷Ⅱ）在石墨电极表面硅烷化以导入氨基（－ＮＨ２）,然后用乙基－（３－二甲基丙基）碳二亚胺盐要卤）ＥＤＣ）关活化剂,将单链ＤＮＡ（共价固定在石墨电极表面。采用显微分光光度法、红外光谱法和电化学方法对电极表面的ｓｓＤＮＡ层进行了表征,并用紫外－可见光谱法对电极表面固定化ｓｓＤＮＡ的杂交特性进行了研究。结果表明,ｓｓＤＮＡ可以比较均匀地固定在石墨电极     

DTPA—Eu^3＋螯合物在核酸杂交分析中的应用

报道了靶ＤＮＡ／生物素化λＤＮＡ探针／亲合素／Ｂｉｏ－ＢＳＡ（ＴＧ－Ｅｕ＾３＋体在核酸杂交分析中的应用，亲合素做为连结杂交体与ｉｏ－ＢＳＡ－ＤＴＰＡ－Ｅｕ＾３＋的桥，生物素和ＤＴＰＡ－Ｅｕ＾３＋均标记在载体蛋白（ＢＳＡ，ＴＧ）上，优化了分析条件，比较了己二胺，乙二胺转胺化探针及载体蛋白对杂交分析的影响，方法灵敏度高，重现性较好（ＲＳＤ－５．７％，ｎ＝６）。     

化学发光核酸传感器的研制

用Ｎ－乙酰半胱氨酸金表面自组装技术,以及用１－乙基３－（３－二氨基）碳二亚胺盐酸盐（ＥＤＣ）、Ｎ－羟基硫代琥珀酰亚胺（ＮＨＳ）偶联剂把亲合素固定于金表面,再将生物素标记探针固定在亲合素上,来制备核酸传感器探头。然后将探头与目标ＤＮＡ和第二生物素标记探 针进行夹心式杂交,再利用生物素－亲合素的作用联入亲合素标记的碱性磷酸酯酶,用酶催化底物ＡＭＰＰＤ发光来达到测定ＤＮＡ的目的。测定也乙型肝炎病毒（ＨＢ     

核酸分子嵌入剂

求文对核酸分子嵌入剂在核酸的识别、分离及分析中的应用，嵌入剂在特效新药设计及对核酸序列的特异位点裂解方面的新成就进行了评述。讨论了嵌入剂与溶液中ＤＮＡ及固定于ＮＣ膜上的单链ＤＮＡ的作用机理，引用参考文献３２篇。     

光子晶体薄膜的制备以及在编码载体中的应用

通过将由聚苯乙烯纳米粒子构成的光子晶体膜镶嵌在聚二甲基硅氧烷(PDMS)薄膜中制备得到了具有PDMS/光子晶体/PDMS夹心结构的可用于多重生物分析的光子晶体编码载体. 用编码载体进行了大肠杆菌3种基因的杂交检测: 以3种光子晶体膜作为编码载体固定核酸探针, 然后在含有荧光标记的目标分子的缓冲液中进行杂交反应. 杂交反应后以光子晶体膜的特征反射谱为核酸编码, 以荧光信号的有无来确定目标分子的存在与否. 实验结果表明PDMS/光子晶体/PDMS夹心结构是一种有效的构建悬浮载体的方法.     

DTPA－Eu~（3＋）螯合物在核酸杂交分析中的应用

报道了靶ＤＮＡ／生物素化λＤＮＡ探针／亲合素／Ｂｉｏ－ＢＳＡ（ＴＧ）－ＤＴＰＡ－Ｅｕ~（３＋）体系在核酸杂交分析中的应用。亲合素做为连结杂交体与Ｂｉｏ－ＢＳＡ－ＤＴＰＡ－Ｅｕ~（３＋）或Ｂｉｏ－ＴＧ－ＤＴＰＡ－Ｅｕ~（３＋）的桥，生物素和ＤＴＰＡ－Ｅｕ~（３＋）均标记在载体蛋白（ＢＳＡ，ＴＧ）上。优化了分析条件，比较了己二胺、乙二胺转胺化探针及载体蛋白对杂交分析的影响。方法灵敏度高（１ｐｇ／ｗｅｌｌ靶λＤＮＡ），重现性较好（ＲＳＤ＝５．７％，ｎ＝６）。     

新型超支状液晶核酸传感器用于p53突变基因的检测

基于核酸杂交链式反应影响液晶取向的原理, 构建了一种新型的超支状液晶核酸传感器用于检测p53突变基因. 本文突破传统构建超支状分子的方式, 采用杂交链式反应方法, 以目标序列p53突变基因作为引发剂, 3种不同的发卡探针Hairpin A, Hairpin B和Hairpin C为单体, 在温和的条件下, 通过改变单体的浓度和反应时间自发杂交组装形成尺寸和分子量可控的超支状DNA(branched-like DNA, bDNA). 借助捕获探针将该超支状DNA连接到液晶传感基底表面, 观察液晶分子取向改变前后的光学信号, 实现了p53基因含249密码子突变序列的快速检测. 本方法有望为核酸诊断的发展提供一种新的方法和思路.     

Glassy carbon electrode modified with gold nanoparticles and DNA for the simultaneous determination of uric acid and norepinephrine under coexistence of ascorbic acid.

A novel biochemical sensor was assembled on a glass carbon electrode by a electrodeposition process. Gold nanoparticles were deposited by potential +1.5 V on the glass carbon electrode. CT-DNA was deposited by potential +1.5 V on the gold nanoparticles array electrode. The properties of the modified electrode were characterized by electrochemical impedance spectroscopy (EIS), X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). Our results showed that gold nanoparticles increase the DNA membrane capacitance and reduce the membrane resistance. This modified electrode can selectively determine uric acid and norepinephrine in the presence of a larger amount of ascorbic acid. The fabricated electrode showed good sensitivity, reproducibility, and stability.     

Development of a Novel Electrochemical Sensor Based on Functionalized Carbon Black for the Detection of Guanine Released from DNA Hydrolysis

In this work, we reported a new strategy based on DNA strands hydrolysis in combination with the employment of functionalized carbon black modified screen-printed electrode (f-CB/SPE) for guanine detection. Using glass microbeads as a solid substrate of ssDNA probe fixation, and under appropriate conditions of temperature, time, and hydrochloric acid concentration, DNA hydrolysis has been successfully used to release the guanine, allowing its oxidation at f-CB/SPE using differential pulse voltammetry. The selectivity was successfully tested with a non-complementary sequence, and a detection limit of 0.7 nM of the target DNA has been reached.     

Fluorescence detection of DNA by the catalytic activation of an aptamer/thrombin complex

A conjugate consisting of a thrombin aptamer tethered to the thrombin, Th, with a sensing nucleic acid (1) is used for the optical detection of DNA. The thrombin/aptamer complex blocks the biocatalytic functions of Th. Hybridization of the analyte DNA (2) to the sensing nucleic acid 1 yields a rigid duplex that detaches the aptamer from Th, a process that activates the protein toward the hydrolysis of bis(p-tosyl-Gly-Pro-Arg)-R110 (3) to the rhodamine 110 fluorophore (4). The system allows the DNA sensing with a sensitivity limit of 1 x 10-8 M. The aptamer/Th conjugate is also immobilized on glass slides for the optical detection of DNA. The dissociation of the aptamer/Th complex upon hybridization and the subsequent dehybridization of the duplex and the regeneration of the catalytically inactive Th/aptamer complex duplicate machinery functions.     

Oxidative polymerization of aniline on the surface of silica in the presence of poly(sulfonic acids) as a method of preparing efficient biosorbents

Polyaniline coatings on the surface of the macroporous silica have been prepared by oxidative polymerization via protonation of aniline by poly(sulfonic acids): poly(p,p′-(2,2′-disulfonic acid)diphenyle-neisophthalamide and poly(p,p′-(2,2′-disulfonic acid)diphenyleneterephthalamide. Two variants of modification have been studied: namely, the polymerization of aniline carried out in the presence of the preliminarily silaminated glass covered by poly(sulfonic acid) and the modification of silaminated glass by preformed complexes of poly(p,p′-(2,2′-disulfonic acid)diphenylenephthalamide-polyaniline. In both cases, the even polyaniline-containing polymer coating with a thickness of ～3 nm is formed on the support surface. Sorbents containing the poly(p,p′-(2,2′-disulfonic acid)diphenylenephthalamide-polyaniline complex are efficient for isolation of DNA from mixtures of biopolymers.     

Stretching of single DNA molecules complexed with restriction endonuclease by Langmuir-Blodgett method

We have proposed a new technique for stretching single double-stranded DNA molecules on solid substrates by the Langmuir–Blodgett (LB) method. The polyion complex monolayer of a cationic amphiphile and DNA molecules formed at the air–water interface was transferred on a clean glass substrate. Vertical lifting up of the glass substrate provided the transferred monolayer consisting the stretched individual DNA molecules aligned parallel to the lifting direction on the glass. The DNA molecules complexed with the restriction endonuclease (EcoRI) were employed for stretching by using this method. Fluorescence images of the transferred monolayer showed that the EcoRI-binding DNA molecules could be stretched and immobilized on the glass substrate. A specific sequence of DNA recognized by EcoRI was detected as spatial positions of the stretched DNA molecules.     

Molecular interactions between DNA and an aminated glass substrate

With the development of DNA arrays, the immobilization of DNA strands onto solid substrates remains an essential research topic. DNA arrays have potential applications in DNA sequencing, mutation detection, and pathogen identification. DNA bound to solid substrates must still be accessible and retain the ability to hybridize with its complementary strands. One technology to produce these arrays involves linking DNA molecule probes to a silanized substrate in microspot patterns and exposing them to a solution of fluorescently labeled samples of DNA targets. The behavior of both the target and probe DNA and their interactions with each other at the substrate surface, particularly with respect to molecular interactions, are poorly understood at the present time. The objective of this work is to model simply the interface interactions between DNA and glass slides modified with an aminosilane (gamma-aminopropyltriethoxysilane, APTS). In aqueous solutions, DNA behaves as a polyacid over a wide range of pH. A glass substrate treated with APTS is positively or negatively charged, depending on the pH. A model of the surface charge of APTS-treated glass has been developed from results of wetting experiments performed at various pH. It has been demonstrated that the surface charge of APTS-treated glass is well described by a model of constant capacitance of the electrical double layer. A good correlation between experimental data on DNA retention at various pH's and the variation of the surface charge of the APTS-treated glass is obtained. This provides an indication of the role of ionic interactions in the adsorption of DNA molecules onto aminated glass slides.     

A simple DNA Characterization method using fiber-fluorescence in situ hybridization performed without DNA fragmentation

We performed high-resolution fluorescence imaging of lambda phage DNA molecules hybridized with fluorescent-labeled DNA and peptide nucleic acid probes. In this method, the target DNA and probe were mixed, rapidly denatured and then subjected to liquid hybridization conditions. The hybridized DNA sample was then spotted onto a nontreated glass substrate and subjected to molecular combing. The resultant continuous fluorescence signal of intact lambda DNA shows that the fluorescent-labeled probes bound to the predicted sites but in a pattern that was clearly different to the beads-on-a-string pattern typical for fiber-fluorescence in situ hybridization. The key changes to the conventional method are hybridization of the free target DNA in liquid and lowering the denaturation temperature. The method described here allows the rapid and direct visualization of the specific binding sites of intact DNA molecules without damaging the DNA fibers and causing fragmentation of the fluorescence signal. This technique should be a useful tool in studies of genetics and also large-scale DNA sequencing projects.     

Fabrication of DNA polymer brush arrays by destructive micropatterning and rolling-circle amplification

A method for fabricating DNA polymer brush arrays using photolithography and plasma etching followed by solid-phase enzymatic DNA amplification is reported. After attaching oligonucleotide primers to the surface of a glass coverslip, a thin layer of photoresist is spin-coated on the glass and patterned via photolithography to generate an array of posts in the resist. An oxygen-based plasma is then used to destroy the exposed oligonucleotide primers. The glass coverslip with the primer array is assembled into a microfluidic chip and DNA polymer brushes are synthesized on the oligonucleotide array by rolling-circle DNA amplification. We have demonstrated that the linear polymers can be rapidly synthesized in situ with a high degree of control over their density and length.     

Site-selective immobilization of gold nanoparticles functionalized with DNA oligomers

The organization of metal and semiconductor nanoparticles to form micro- and nanostructured assemblies is currently of tremendous interest. This communication reports on the utilization of DNA molecules as positioning elements for generating microstructured surface architecture from gold nanoparticles. Citrate-passivated 40 nm gold colloids were modified by chemisorptive coupling with a 5′-thiol-derivatized DNA oligomer. The nucleic acid was used as a molecular handle for the specific immobilization on solid supports, previously functionalized with capture DNA oligomers, complementary to the nanoparticle-bound DNA. As a consequence of the enormous specificity of nucleic acid hybridization, the DNA-directed immobilization (DDI) allows, to site-specifically target the hybrid nanoparticles to microlocations which contain the complementary oligomers. The site-selectivity of the surface adsorption is demonstrated by immobilizing the gold colloids on a DNA microarray on a glass cover slide. Moreover, scanning force microscopy (SFM) analysis, used to characterize the intermediate steps of the DDI on a gold substrate, provided initial insights into the specificity and efficiency of this technique. The application of the DDI to fabricate complex colloidal micro- and nanostructures is anticipated. Received: 26 July 2000/Accepted: 5 October 2000