DNA微阵列制备的新进展

随着人类基因组测序的完成和蛋白组学工程的开展，具有高产出测序的特性的DNA微阵列技术的发展日新月异，其应用已经深入到了生命科学研究的很多方面．与此同时，几种新的DNA微阵列制备技术迅速发展起来，文章介绍了滚轮放大技术、聚酰胺胺表面法、三甲氧基对胺苯基硅烷／重氮化法、化学纳米印迹法等四种制备方法的新进展。     

X-ray photoelectron spectroscopic investigations of modifications in plasmid DNA after interaction with Hg nanoparticles

The electronic structure of plasmid DNA both prior to and after its conjugation with Hg nanoparticles (NP) has been investigated here. The DNA molecules undergo severe perturbation on their interaction with Hg-NP. The circular plasmid DNA becomes predominantly linearised on interaction with 10 mM Hg salt solution. These perturbations are also reflected in the electronic structure of C1s and O1s suggesting severe modifications in the exocyclic groups, of the nucleic acid bases, participating in the hydrogen bondings. The results reflect the rupturing of the purine-pyrimidine bonds after interaction with the NP. The study also suggests selective and specific interactions of DNA bases with the Hg nanoparticles leading to the formation of metal-purine-pyrimidine complexes through rupturing of G-C base pairs. Hg-NP exclusively interact with the nitrogen bases of the DNA molecule and do not cause any significant modification to the phosphate backbone of DNA.     

DNA折纸结构介导的多尺度纳米结构精准制造

原子及近原子尺度制造在近年来一直是物质科学领域被广泛探讨的前沿问题.当制造和加工的尺度从微米、纳米逐渐走向原子级别时,材料在常规尺度下所具备的性质已无法通过经典理论进行解释,相反地,会在这一尺度下展现出一系列新奇的特性.因而对材料极限制造尺度和颠覆性物性的不断追求始终是科学界共同关注的重点领域.作为一种在纳米尺度下对结构制造单元进行精细操控的先进手段,DNA纳米技术的开发和发展为纳米制造甚至原子制造提供了新的观点和思路,而DNA折纸术作为DNA纳米技术的重要组成部分,正在凭借其在结构制造过程当中的高度可编程性成为纳米尺度下进行各类物质精准制造的独特的解决方案,并可能为不同物质不同材料更小尺度和任意形状的精准构筑带来机遇.本文首先简单概述了DNA折纸术的基本原理和发展历程,然后根据制造策略的不同对DNA折纸结构的纳米制造的相关代表性工作做了总结,并在文末提出了对于DNA折纸结构在原子制造中的可行性的思考和未来发展方向的展望.     

Regulation of the elastic modulus of polyurethane microarrays and its influence on gecko-inspired dry adhesion

We studied the influence of the elastic modulus on the gecko-inspired dry adhesion by regulating the elastic modulus of bulk polyurethane combined with changing the size of microarrays. Segmented polyurethane (PU) was utilized to fabricate micro arrays by the porous polydimethyl siloxane (PDMS) membrane molding method. The properties of the micro arrays, such as the elastic modulus and adhesion, were investigated by Triboindenter. The study demonstrates that bulk surfaces show the highest elastic modulus, with similar values at around 175 MPa and decreasing the arrays radius causes a significant decrease in E, down to 0.62 MPa. The corresponding adhesion experiments show that decrease of the elastic modulus can enhance the adhesion which is consistent with the recent theoretical models.     

Optimization of Gold Nanoparticle-Based DNA Detection for Microarrays

DNA microarrays are promising tools for fast and highly parallel DNA detection by means of fluorescence or gold nanoparticle labeling. However, substrate modification with silanes (as a prerequisite for capture DNA binding) often leads to inhomogeneous surfaces and/or nonspecific binding of the labeled DNA. We examined both different substrate cleaning and activating protocols and also different blocking strategies for optimizing the procedures, especially those for nanoparticle labeling. Contact angle measurements as well as fluorescence microscopy, atomic force microscopy (AFM), and a flatbed scanner were used to analyze the multiple-step process. Although the examined different cleaning and activating protocols resulted in considerably different contact angles, meaning different substrate wettability, silanization led to similar hydrophobic surfaces which could be revealed as smooth surfaces of about 2–4 nm roughness. The two examined silanes (3-glycidoxypropyltrimethoxysilane (GOPS) and 3-aminopropyltriethoxysilane (APTES)) differed in their DNA binding homogeneity, maximum signal intensities, and sensitivity. Nonspecific gold binding on APTES/PDC surfaces could be blocked by treatment in 3% bovine serum albumin (BSA).     

基于DNA自组装的金属纳米结构制备及相关纳米光子学研究

纳米光子学是研究光在纳米尺度下的行为以及光和纳米材料相互作用的一门科学.金属纳米材料凭借其独特的表面等离子体效应,可以在衍射极限以下对光进行传递和聚焦,因而是纳米光子学研究的重点.大量研究表明,通过调控金属纳米材料的形貌和成分可以控制表面等离子体的性质,从而对光进行可控调节.近年来,随着DNA纳米技术的发展,又为纳米光子学的发展带来了新的机遇.首先,人们发现不同的DNA序列可以调控金属纳米颗粒的成长,从而影响金属纳米颗粒的形貌和成分.此外,利用DNA自组装技术,可以将金属纳米颗粒组装成为有序可控的纳米结构.因此,基于DNA的纳米光子学研究近年来发展十分迅速.在此背景下,本文对相关研究进行归纳与总结,以期吸引更多研究人员的关注,推动该领域的进一步发展.本文首先介绍了金属纳米结构基于表面等离体实现突破光学衍射极限的原理,然后按照DNA对金属纳米结构的形貌或成分影响方式的不同分成若干部分,对基于DNA的纳米光子学做了系统的综述,最后展望了未来可能的发展方向.     

Printing technologies for fabrication of bioactive and regular microarrays of streptavidin

In this study, we report and compare two methods for fabricating patterns of streptavidin protein using soft litography microprinting technique (μCP) and laser-based method termed ‘matrix assisted pulsed laser evaporation direct write’ (MAPLE DW). The μCP approach is a parallel deposition technique capable of X depositions per stamper. The technique is limited in more sophisticated multicomponent deposition by the size of patterns that can be produced and the features obtained during the transfer process. The computer-aided design/computer-aided manufacturing (CAD/CAM) ability of MAPLE DW overcomes the limitations of the μCP approach. (i) We establish the science and engineering principles behind the effective transfer of microarrays and (ii) we explore issues regarding the direct immobilization, morphology and function of the deposited protein at the interface with an aqueous environment and in the precision of controlled ligand-receptor reactions. In summary, our objective was to develop simple, robust microfabrication techniques for the construction of model 2D and 3D bioscaffolds to be used in fundamental bioengineering studies.     

Mass fabrication of size-controllable hydrogel microarrays by dip-pen nanolithography with viscosity-tunable ink

A method of mass fabricating poly(ethylene glycol) (PEG) hydrogel microarrays is demonstrated. Microarrays of poly(ethylene glycol) dimethacrylate (PEG-DMA) with photoinitiator were patterned by one-dimensional (1-D) parallel dip-pen nanolithography (DPN), and the microarrays were cross-linked to form PEG hydrogels by UV irradiation in N₂ air. As an ink material for DPN printing, solid and liquid phase of PEG-DMA were mixed and prepared to tune viscosity of the ink material by temperature. Thus, the diameter of the microarrays was able to be averagely controlled from 1.7 to 6.2 μm as temperature during printing was increased from 25 °C to 37 °C, respectively. The overall microarrays showed less than 16% coefficient of variation (C.V.). Moreover, small molecules, such as fluorescence dyes, were able to be embedded in the PEG hydrogel microarrays.     

An atomic force microscopy study of DNA hairpin probes monolabelled with gold nanoparticle: Grafting and hybridization on oxide thin films

First and original results are reported regarding the surface evolution of two kinds of oxide film after covalent grafting and hybridization of hairpin oligonucleotide probes. These hairpin probes were monolabelled with a 1.4 nm gold nanoparticle. One kind of oxide film was rough Sb doped SnO₂ oxide film and the other kind was smooth SiO₂ film. Same process of covalent grafting, involving a silanization step, was performed on both oxide surfaces. Atomic force microscopy (AFM) was used to study the evolution of each oxide surface after different steps of the process: functionalization, probe grafting and hybridization. In the case of rough SnO₂ films, a slight decrease of the roughness was observed after each step whereas in the case of smooth SiO₂ films, a maximum of roughness was obtained after probe grafting. Step height measurements of grafted probes could be performed on SiO₂ leading to an apparent thickness of around 3.7 ± 1.0 nm. After hybridization, on the granular surface of SnO₂, by coupling AFM with SEM FEG analyses, dispersed and well-resolved groups of gold nanoparticles linked to DNA duplexes could be observed. Their density varied from 6.6 ± 0.3 × 10¹⁰ to 2.3 ± 0.3 × 10¹¹ dots cm⁻². On the contrary, on smooth SiO₂ surface, the DNA duplexes behave like a dense carpet of globular structures with a density of 2.9 ± 0.5 × 10¹¹ globular structures cm⁻².     

Study on the interactions between anti-cancer drug oxaliplatin and DNA by atomic force microscopy

Oxaliplatin is one of the most important anticancer drugs at present. However, the mechanism of action of oxaliplatin is still controversial. In this study, the interactions between oxaliplatin and a plasmid DNA have been studied so as to reveal the structural basis of its activity. The structural characteristic of pUC19 DNA (2 ng/μL) incubated with 100 μmol/L and 1000 μmol/L of oxaliplatin for the different time on a freshly cleaved highly ordered pyrolytic graphite (HOPG) surface was investigated by atomic force microscopy (AFM). High resolution AFM observation indicated that oxaliplatin can induce pUC19 DNA molecules change from the extended conformation to the entangled structures with many nodes, and finally to the compact particles. The present AFM results provide structural evidence about the interactions between oxaliplatin and circular duplex DNA containing multiple targets.     

Mass spectrometric characterization of DNA microarrays as a function of primary ion species

Recent studies have shown TOF-SIMS to be an appropriate method for the detailed examination of the immobilization process of PNA and its ability to hybridize to unlabeled complementary DNA fragments. Unlabeled single-stranded DNA was hybridized to Si wafer biosensor chips containing both complementary and non-complementary immobilized PNA sequences. The hybridization of complementary DNA could readily be identified by detecting phosphate-containing molecules from the DNA backbone. An unambiguous discrimination was achieved between complementary and non-complementary sequences.In order to optimize detection parameters, different primary ions were applied, including monoatomic ions (Bi⁺) as well as cluster ions (Bi₂⁺, Bi₃⁺, Bi₄⁺, Bi₃⁺⁺, Bi₅⁺⁺), and secondary ion yield behavior and formation efficiencies were studied. It was found that cluster primary ions resulted in a significantly increased yield of DNA-correlated fragments, enabling higher signal intensities and better secondary ion efficiencies.TOF-SIMS is undoubtedly a highly useful technique for identifying hybridized DNA on PNA biosensor chips. It is suitable for studying the complexity of the immobilization and hybridization processes and may provide a rapid method for DNA diagnostics. With the absence of the labeling procedure and the simultaneous increase of the phosphate signal as a result of increasing DNA sequence length, this technique comes to be especially useful for the direct analysis of genomic DNA.     

Synthesis, characterization of the pentacene and fabrication of pentacene field-effect transistors

A comprehensive understanding of the organic semiconductor material pentacene is meaningful for organic field-effect transistors (OFETs). Thin films of pentacene are the most mobile molecular films known to date. This paper reported that the pentacene sample was successfully synthesized. The purity of pentacene is up to 95\%. The results of a joint experimental investigation based on a combination of infrared absorption spectra, mass spectra (MS), element analysis, x-ray diffraction (XRD) and atom force microscopy (AFM). The authors fabricated OFET with the synthesized pentacene. Its field effect mobility is about 1.23\,cm$^2$/(V$\cdot$s) and on--off ratio is above 10$^{6}$.     

用液流操纵单个DNA分子形成纳米悬链线图形

首先用液流拉伸单分子DNA,使其吸附在修饰过的云母基片上.然后让二次液流沿着垂直于已拉伸的DNA的方向流过云母片,用原子力显微镜(AFM)观测,可见DNA单分子片段在基片上形成了一些纳米尺度的悬链线.提出一种“S悬桥”模型能定量地解释这一现象.该研究工作揭示,在DNA的单分子操作中,经典的弹性力学理论足以描述和控制DNA分子二维图形的形成 关键词： DNA 单分子DNA操纵 原子力显微镜(AFM) 纳米悬链线     

Synthesis, characterization of the pentacene and fabrication of pentacene field-effect transistors

A comprehensive understanding of the organic semiconductor material pentacene is meaningful for organic fieldeffect transistors （OFETs）. Thin films of pentacene are the most mobile molecular films known to date. This paper reported that the pentacene sample was successfully synthesized. The purity of pentacene is up to 95%. The results of a joint experimental investigation based on a combination of infrared absorption spectra, mass spectra （MS）, element analysis, x-ray diffraction （XRD） and atom force microscopy （AFM）. The authors fabricated OFET with the synthesized pentacene. Its field effect mobility is about 1.23 cm^2/（V·s） and on-off ratio is above 10^6.     

DNA纤维的X射线衍射分析与双螺旋结构的发现

概述了DNA化学结构式确立的过程,介绍了x射线衍射分析的概念、原理和方法,阐述了物理学家威尔金斯和物理化学家富兰克林在用实验事实证明DNA双螺旋结构方面所作出的巨大贡献。     

Immobilization of redox mediators on functionalized carbon nanotube: A material for chemical sensor fabrication and amperometric determination of hydrogen peroxide

Chemical functionalization of single-walled carbon nanotubes with redox mediators, namely, toluidine blue and thionin have been carried out and the performance of graphite electrode modified with functionalized carbon nanotubes is described. Mechanical immobilization of functionalized single-walled nanotube (SWNT) on graphite electrode was achieved by gently rubbing the electrode surface on carbon nanotubes supported on a glass slide. The electrochemical behaviour of the modified electrodes was investigated by cyclic voltammetry. The SWNT-modified electrodes showed excellent electrocatalytic effect for the reduction of hydrogen peroxide. A decrease in overvoltage was observed as well as an enhanced peak current compared to a bare graphite electrode for the reduction of hydrogen peroxide. The catalytic current was found to be directly proportional to the amount of hydrogen peroxide taken.     

“Versatile toolset” for DNA or protein immobilization: Toward a single-step chemistry

Covalent immobilization of non-modified biological materials as proteins or nucleic acids has been performed through a single and soft method. Based on diazonium salt chemistry, this protocol leads to an ultrathin grafted film, on metallic or polymer materials, which can eventually be used as a self-adhesive primer for immobilizing biological materials from aqueous solutions through a simple dipping step. Moreover, this self-adhesive primer may be patterned by cheap and easy methods as ink or UV masking. Biological models as low molecular weight DNA from salmon sperm and glucose oxidase (GOD) were covalently immobilized by this soft procedure. In order to evaluate the consequences of this non-specific covalent immobilization method on biological activity, enzymatic activity of GOD was monitored by electrochemical detection of hydrogen peroxide (H₂O₂). We thus demonstrate that such a self-adhesive primer represents a new and alternative process offering a versatile toolset for immobilizing biological material for biosensor development on conductive and non-conductive materials.     

Effect of fabrication parameters on morphological and optical properties of highly doped p-porous silicon

Porous silicon (PS) layers were fabricated by anodization of low resistive (highly doped) p-type silicon in HF/ethanol solution, by varying current density, etching time and HF concentration. Atomic force microscopy (AFM) and field emission scanning electron microscope (FESEM) analyses were used to investigate the physical properties and reflection spectrum was used to investigate the optical behavior of PS layers in different fabrication conditions. Vertically aligned mesoporous morphology is observed in fabricated films and with HF concentration higher than 20%. The dependence of porosity, layer thickness and rms roughness of the PS layer on current density, etching time and composition of electrolyte is also observed in obtained results. Correlation between reflectivity and fabrication parameters was also explored. Thermal oxidation was performed on some mesoporous layers that resulted in changes of surface roughness, mean height and reflectivity of the layers.     

桑色素合镧(Ⅲ)与核酸作用的荧光光谱及其分析应用

采用荧光法研究了桑色素合镧 (Ⅲ )配合物与核酸的作用。研究了溶液的pH值、缓冲溶液含量、有机试剂、外加干扰离子对桑色素合镧 (Ⅲ )配合物与核酸体系荧光光谱的影响 ,同时还研究了变性DNA与桑色素合镧 (Ⅲ )配合物之间的作用。在溶液 pH =8 0～ 9 0的条件下 ,体系的荧光强度最大 ,最大激发波长为387nm ,最大发射波长为 5 35nm。缓冲溶液的用量控制在 10 % (体积 ) ,乙醇用量也控制在 10 % (体积 )。配合物荧光强度在核酸存在时有所增加 ,据此确定了测定核酸的一种方法 ,简便、快速 ,线性范围宽 ,灵敏度较高 ,结果准确。并对可能的增色效应和酸度对荧光光谱影响的原因进行了讨论。     

Labeling of cytosine residues with biliproteins for use as fluorescent DNA probes

The fluorescent properties of biliproteins (B-phycoerythrin, BPE; C-phycocyanin, CPC and allophycocyanin, APC) have been utilized as labels of nucleic acids to detect hybridization by means of steady-state fluorescence anisotropy in a homogeneous aqueous solution of model system in which poly(C) and poly(I) are, respectively, the probe and target sequences. An easy method to obtain biliprotein-DNA conjugates by a two-stage procedure is described. The first stage was a modification of the cytosine amino group of poly(C) at the N⁴ position which then reacted with N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) to obtain poly(C)-bound 2-pyridyl disulfide. In the second stage biliproteins were directly reacted with SPDP to obtain biliprotein-bound 2-pyridyl disulfide, dithiotreitol was added for reduction to biliprotein-bound thiol and was then mixed with the poly(C)-bound 2-pyridyl disulfide to obtain the biliprotein-poly(C) conjugate. The three biliproteins studied bind to nucleic acids without noticeable change of their spectral properties (absorption, fluorescence efficiency and fluorescence lifetime). Consequently, our labeling methodology can be applied to obtain any type of biliprotein-labeled nucleic acid probe. The increase of the steady-state fluorescence anisotropy from biliprotein-poly(C) upon hybridization with poly(I) can be used to readily detect the hybridization with the target poly(I) in a sample without having to separate free and bound labeled probes. The small decreases in lifetime displayed by the biliprotein-poly(C) conjugates upon hybridization are not sufficient to explain the steady-state anisotropy increase. Apparently, the rotational motion of the overall macrostructure is principally responsible for the increase in anisotropy. The greater fluorescence efficiency and lifetime from BPE with respect to those from CPC or APC allows us recommend that protein as the most suitable label.