A nanogold-quenched fluorescence duplex probe for homogeneous DNA detection based on strand displacement

A nanogold-quenched fluorescence duplex probe has been developed for lighting up homogenous hybridization assays. This novel probe is constructed from two strands of different lengths, and labeled by nanogold and a fluorophore at the long-strand 5′-end and the short-strand 3′-end, respectively. The two tags are in close contact, resulting in complete quenching of the probe fluorescence. If perfectly complemented to the nanogold-labeled strand, a long target oligonucleotide would displace the short fluorophore-labeled strand, and as a result, restore the fluorescence. By using nanogold in the probe, an extremely high quenching efficiency (99.1%) and removal of free fluorophore-labeled strand is achieved. The signal-to-noise ratio and the detection limit (50 pmol L⁻¹) of homogenous assays are therefore improved significantly, in comparison with similar probes using organic acceptors. Moreover, the probe has a great inhibition effect on hybridization to a mismatched oligonucleotide. This effect provides the assay with a high specificity, and particularly the assay has great potential in applications for discriminating variations in sequences. The assay sensitivity could be markedly enhanced by using fluorescent materials in the signal strand that are brighter and not quenched by nucleobases.     

Gold nanoparticle aggregation-based highly sensitive DNA detection using atomic force microscopy

The potential ability of atomic force microscopy (AFM) as a quantitative bioanalysis tool is demonstrated by using gold nanoparticles as a size enhancer in a DNA hybridization reaction. Two sets of probe DNA were functionalized on gold nanoparticles and sandwich hybridization occurred between two probe DNAs and target DNA, resulting in aggregation of the nanoparticles. At high concentrations of target DNA in the range from 100 nM to 10 μM, the aggregation of gold nanoparticles was determined by monitoring the color change with UV-vis spectroscopy. The absorption spectra broadened after the exposure of DNA–gold nanoparticles to target DNA and a new absorption band at wavelengths >600 nm was observed. However, no differences were observed in the absorption spectra of the gold nanoparticles at low concentrations of target DNA (10 pM to 10 nM) due to insufficient aggregation. AFM was used as a biosensing tool over this range of target DNA concentrations in order to monitor the aggregation of gold nanoparticles and to quantify the concentration of target DNA. Based on the AFM images, we successfully evaluated particle number and size at low concentrations of target DNA. The calibration curve obtained when mean particle aggregate diameter was plotted against concentration of target DNA showed good linearity over the range 10 pM to 10 nM, the working range for quantitative target DNA analysis. This AFM-based DNA detection technique was three orders of magnitude more sensitive than a DNA detection method based on UV-vis spectroscopy.     

非标记电化学DNA杂交指示剂

电化学DNA杂交检测技术因其快速、灵敏、低消耗和易于操作等优点而在临床医学、环境监测和药物分析等领域受到普遍关注。电化学DNA杂交指示剂是DNA电化学杂交传感器的重要组成部分,能与单链DNA和双链DNA通过不同的模式和作用力进行差异性结合。本文介绍了电化学杂交指示剂的定义及其在DNA电化学传感器中的重要性,根据分子结构上的特征差异,将非标记型电化学杂交指示剂分为有机染料（荧光素）、药物分子和金属配合物三类,并从中选取了各个类型中具有代表性的指示剂对它们的工作原理、研究进展和应用现状进行了比较和评述。对杂交指示剂的设计和开发前景,特别是满足基因芯片应用等方面做出了展望。     

电化学发光分析的新进展

电化学发光技术作为一种新的痕量生化分析手段越来越引人注目。介绍了电化学发光基础研究的最新进展,并评述了电化学发光技术的环境分析,免疫分析,核酸杂交分析及其它生化物质测定中的一些最新应用。     

Nondoped blue fluorescent OLED based on cyanophenanthrimidazole‐styryl‐triphenylamine/carbazole materials

New 2‐(4′‐9H‐carbazole‐9‐yl)‐styryl‐1H‐phenathro[9,10‐d]imidazole‐1‐yl)benzonitrile (SPICN‐Cz) and 4‐(2‐(4‐(diphenylamino)phenyl‐styryl‐1H‐phenathro[9,10‐d]imidazole‐1‐yl)benzonitrile (SPICN‐TPA) have been synthesised, and their photophysical, electrochemical, and electroluminescent properties were analysed in comparison with their cyano‐free parent compounds, SPI‐Cz, and SPI‐TPA. Solvatochromic effects show the transformation of an excited state character from locally excited (LE) state to charge transfer (CT) state. Using time‐dependent density functional theory calculation, the excited state properties of these donor‐acceptor blue emissive materials have been analysed. Their excited state properties have been tuned by replacing the strong donor triphenylamine to weak donor carbazole to achieve the combination of high photoluminance efficiency locally excited (LE) component and high exciton‐utilizing CT component in one excited state. Hybridization processes between LE and CT components of SPICN‐Cz and SPICN‐TPA in the emissive state have been discussed. The nondoped organic light emitting diode device based on SPICN‐Cz exhibit better electroluminescent performances than those of SPICN‐TPA–based device: high external quantum efficiency of 2.58 %, current efficiency of 2.90 cd A^‐1, and power efficiency of 2.26 lm W^‐1 with Commission Internationale de l'Éclairage (CIE) coordinates of (0.15, 0.12). The excited state modulation and the composition of LE and CT states in the donor‐acceptor system could be useful to design low‐cost, high‐efficiency fluorescent organic light emitting diode materials.     

基于纳米金探针和基因芯片的DNA检测新方法

运用荧光纳米金探针和基因芯片杂交建立一种新的DNA检测方法. 荧光纳米金探针表面标记有两种DNA探针: 一种为带有Cy5荧光分子的信号探针BP1, 起信号放大作用; 另一种为与靶DNA一部分互补的检测探针P532, 两种探针比例为5∶1. 当靶DNA存在时, 芯片上捕捉探针(与靶DNA的另一部分互补)通过碱基互补配对结合靶DNA, 将靶DNA固定于芯片上; 荧光纳米金探针通过检测探针与靶DNA及芯片结合, 在芯片上形成“三明治”复合结构, 最后通过检测信号探针上荧光分子的信号强度来确定靶DNA的量. 新方法检测灵敏度高, 可以检测浓度为1 pmol/L的靶DNA, 操作简单, 检测时间短. 通过改进纳米金探针的标记和优化杂交条件, 可进一步提高核酸检测的灵敏度, 这将在核酸检测方面具有重要的应用价值.     

Application of peptide nucleic acids containing azobenzene self-assembled electrochemical biosensors in detecting DNA sequences

Hybridization of peptide nucleic acids probe containing azobenzene (NH₂-TNT₄, N-PNAs) with DNA was performed by covalently immobilizing of NH₂-TNT₄ in sequence on the 3-mercaptopropionic acid self-assembled monolayer modified gold electrode with the helps of N-(3-dimethylaminopropy1)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and the hybrid was coded as N-PNAs/DNA. Using [Fe(CN)₆]^4−/3− (1:1) as the electrochemical indicator, the electrochemical properties of the N-PNAs self-assembled monolayer (N-PNAs-SAMs) and N-PNAs/DNA hybridization system under the conditions of before and after UV light irradiation were characterized with cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectra (EIS). Results showed that the redox currents decreased with the increase of irradiation time, suggesting that the ability of the charge transfer on the electrode surface was weakened and the conformation of hybrid system had been changed, and the control of PNAs/DNA hybridization could be realized by UV light irradiation. Supported by the National Natural Science Foundation of China (Grant No. 50572107) and “Top Hundred Talents Program” of Chinese Academy of Science     

Label-free detection of DNA mutations by SPR: application to the early detection of inherited breast cancer

A screening analysis of DNA hybridization and the presence of DNA mutations using an surface plasmon resonance (SPR) biosensor is shown. The influence of lateral and vertical spacers, as well as several hybridization conditions, was studied to optimize the differentiation between fully complementary and mismatched DNA strands. Our results demonstrated that SPR biosensors were able to detect mismatch sequences related to inherited breast cancer, with high specificity and sensitivity. Using PCR synthetic sequences as targets, mutant sequences were clearly discriminated from fully complementary ones, and detection limits below 50 nM were achieved.     

Twin probes as a novel tool for the detection of single-nucleotide polymorphisms

Single-nucleotide polymorphisms (SNPs) are the most common form of DNA sequence variation. There is a strong interest from both academy and industry to develop rapid, sensitive and cost effective methods for SNP detection. Here we report a novel structural concept for DNA detection based on fluorescence dequenching upon hybridization. The so-called "twin probe" consists of a central fluorene derivative as fluorophore to which two identical oligonucleotides are covalently attached. This probe architecture is applied in homogeneous hybridization assays with subsequent fluorescence spectroscopic analysis. The bioorganic hybrid structure is well suited for sequence specific DNA detection and even SNPs are identified with high efficiency. Additionally, the photophysical properties of the twin probe were investigated. The covalent attachment of two single stranded oligonucleotides leads to strong quenching of the central fluorescence dye induced by the nucleobases. The twin probe is characterized by supramolecular aggregate formation accompanied by red-shifted emission and broad fluorescence spectra.