非标记型p53抑癌基因电化学DNA生物传感器的研究

基于发夹型核酸探针的高特异性识别能力以及电活性物质与DNA磷酸骨架间的静电作用,以发夹型核酸作为分子识别探针,电活性物质六氨合钌(RuHex)作为杂交指示剂,构建了一种非标记型检测p53抑癌基因的电化学DNA生物传感器.实验结果表明,在10 μmol/L RuHex溶液中,该传感器对目标DNA具有灵敏的电化学响应,电化...     

采用电活性标记物N-(2-乙基-二茂铁)马来酰亚胺检测与表面固定的 一致性双链DNA特异性作用的p53蛋白质

建立了电化学检测表面固定捕获的野生型p53蛋白质的方法. 首先在金电极表面形成巯基化的单链DNA探针/己硫醇(HT)混合自组装膜, 随后巯基化的单链DNA探针与溶液中序列匹配的靶点DNA杂交, 所形成的一致性双链DNA捕获溶液中的野生型p53蛋白质. p53分子表面的半胱氨酸残基采用巯基特异性试剂N-(2-乙基-二茂铁)马来酰亚胺(Fc-Mi)进行衍生. 通过检测二茂铁的电化学信号来指示p53与一致性双链DNA之间的特异性相互作用. p53蛋白质与双链DNA的键合程度取决于双链DNA的序列. 该方法可检测的p53最低浓度为1.33 nmol•L－1.     

基于杂交链式反应信号放大和磁分离技术荧光检测端粒酶活性

端粒酶是由RNA和蛋白质组成的一种核糖核蛋白酶, 它一般在癌细胞中被激活. 它与端粒DNA的不断复制以及癌细胞的不断增殖密切相关. 所以检测端粒酶的活性对癌症的早期诊断以及以端粒酶为靶标分子的抗癌药物的开发具有重要意义. 利用杂交链式反应(HCR)无酶放大检测信号, 建立了一种简单、快速的端粒酶活性检测方法. 端粒酶延伸产物是一条末端具有(ggttag)_n重复序列的DNA. 在实验过程中, 通过链霉亲合素与生物素的特异性作用将端粒酶延伸产物连接在磁性微球上. 设计一条端粒酶延伸产物特异性的DNA探针I作为杂交链式反应的引发探针. DNA探针I的3'-端与端粒酶延伸产物的重复序列匹配, 通过杂交, DNA探针I被固定在磁球上; DNA探针I的5'-端引发DNA探针II和探针III发生杂交链式反应. DNA探针II和探针III上都标记有荧光基团, 可以利用荧光直接进行信号检测. 在反应过程中, 通过磁分离去除多余未反应的三种DNA探针. 在优化条件下, 可以检测到1.0×10⁵个Hela细胞中的端粒酶活性. 该方法简单、快速、检测成本低, 分析全程无酶参与, 在肿瘤或癌症的临床诊断以及以端粒酶为靶标分子的抗癌药物的筛选上具有广阔的应用前景.     

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.     

液相表面增强拉曼光谱传感器的制备方法及其核酸检测的应用

本发明公开了液相表面增强拉曼光谱传感器的制备方法及其核酸检测的应用。该传感器包含检测基底和SERS探针两部分。检测基底为四面体DNA探针修饰的磁核枝杈状金壳纳米颗粒,SERS探针为表面修饰有能与目标核酸杂交的特定碱基序列和拉曼信号分子的金纳米颗粒。检测是,将检测基底、SERS探针与待检测液体样品混合,通过碱基互补配对形成"检测基底目标核酸SERS探针"夹心结构复合物,借助外加磁场分离检测液中的复合物并富集后进行SERS测试,利用SERS信号实现了对于血清中核酸的高灵敏、特异性检测,检测限达到f M,可实现在血清等复杂环境中检测核酸标志物。     

基于银纳米粒子构建荧光传感平台用于核酸检测

报道了基于银纳米粒子构建的荧光传感平台,并用于核酸检测.此荧光传感平台对核酸检测基于以下策略:首先,荧光团标记的单链DNA探针被吸附到银纳米粒子的表面,荧光团与银纳米粒子近距离接触,发生荧光猝灭;加入与探针DNA序列互补的目标DNA,两者杂交形成双链DNA,并从银纳米粒子的表面脱离,荧光得到恢复.这种银纳米粒子构建的荧...     

基于双酶切级联信号放大的核酸检测方法

利用茎环结构定位探针构建了一个基于双酶切反应的级联信号放大体系,并将其用于核酸的检测.在该体系中,茎环结构定位探针首先是内切酶Tth EndonucleaseⅣ的作用底物,被剪切后又作为定位探针介导切口酶Nt.Bst NBI对分子信标实施剪切,将这2步剪切反应结合起来可有效克服切口酶对于目标核酸中特定识别序列的依赖,同时进一步提高了检测灵敏度.实验结果表明,荧光信号与目标DNA浓度的对数值呈线性相关,响应范围为1 pmol/L～1 nmol/L,并且具有良好的识别单碱基变异的能力.此外,本方法序列设计简单,通用性强,仅改变定位探针的部分序列即可实现对不同目标DNA的检测.对掺杂于血清中的目标DNA的检测结果验证了本方法在实际样品检测中的应用潜力.     

基于免标记发夹型探针和核酸外切酶Ⅲ的荧光信号放大DNA检测

设计合成了一种长臂发夹型核酸探针,结合核酸外切酶Ⅲ水解反应建立了一种免标记荧光信号放大高灵敏检测DNA的新方法.当不存在靶DNA时,SYBR GreenⅠ荧光染料能够嵌入发夹型探针的茎部而发出很强的荧光,而当存在靶DNA并与发夹型探针杂交后,核酸外切酶Ⅲ从杂交产物的3'端开始水解发夹型探针,释放出靶DNA,并触发下一个酶水解反应,同时SYBR GreenⅠ染料也随发夹型探针水解而释放,导致荧光信号降低,从而实现了对DNA的免标记荧光信号放大高灵敏检测.该方法的检出限低至320 fmol/L,比传统双标的分子信标的方法降低了4~5个数量级,且该方法还具有免标记、简单、快速的特点.     

基因芯片-玻璃表面两种不同DNA探针固定化方法的比较研究

研究了基因芯片相关的DNA探针在芯片表面最佳固定化方法。用两种不同的双功能试剂1,4-苯二异硫氰酸酯和戊二醛分别把5'-端氨基衍生的21-mer寡脱氧核苷酸探针直接共价固定到玻片表面,固定化的寡脱氧核苷酸探针与5'-端FITC标记的互补靶序列进行分子杂交,杂交后用配有CCD的IX70型荧光倒置显微镜成像检测。结果表明,两种固定化方法的效果都比较好,能检测到靶序列的最低终浓度为1.5×10^-9mol/L,优化了探针固定化时间、杂交时间、杂交温度等对DNA芯片分析性能的影响,为构建高灵敏度基因芯片打下良好基础。     

基于核酸适体的新型液晶生物传感用于检测血小板源性生长因子BB

利用核酸适体与其靶分子具有高度特异性结合的原理,建立了基于液晶取向改变的以核酸适体为捕获探针用于检测血小板源性生长因子BB(Platelet-derived growth factor BB,PDGF-BB)分子的新型液晶生物传感方法。核酸适体通过戊二醛偶联固定在3-氨丙基三乙氧基硅烷/N,N-二甲基-N-十八烷基[3-(三甲氧基硅基)丙基]氯化铵[(3-Aminopropyl)trimethoxysilane/N,N-dimethyl-N-octadecyl(3-aminopropyl)trimethoxysilyl chloride,APTES/DMOAP]混合自组装的传感基底表面,当靶分子PDGF-BB存在时,可与核酸适体发生特异性作用结合于传感基底表面,根据生物分子的空间尺寸效应能诱导液晶分子取向发生变化,从而引起光学信号的亮度和色彩发生改变,实现对PDGF-BB的快速检测。本方法具有操作简单、选择型好、灵敏度高的特点,在PDGF-BB浓度为5 nmol/L时仍可观察到明显的光学信号变化。     

Electrochemical quantification of single-nucleotide polymorphisms using nanoparticle probes

We report a new approach for electrochemical quantification of single-nucleotide polymorphisms (SNPs) using nanoparticle probes. The principle is based on DNA polymerase I (Klenow fragment)-induced coupling of the nucleotide-modified nanoparticle probe to the mutant sites of duplex DNA under the Watson-Crick base pairing rule. After liquid hybridization events occurred among biotinylated DNA probes, mutant DNA, and complementary DNA, the resulting duplex DNA helixes were captured to the surface of magnetic beads through a biotin-avidin affinity reaction and magnetic separation. A cadmium phosphate-loaded apoferritin nanoparticle probe, which is modified with nucleotides and is complementary to the mutant site, is coupled to the mutant sites of the formed duplex DNA in the presence of DNA polymerase. Subsequent electrochemical stripping analysis of the cadmium component of coupled nanoparticle probes provides a means to quantify the concentration of mutant DNA. The method is sensitive enough to detect 21.5 attomol of mutant DNA, which will enable the quantitative analysis of nucleic acid without polymerase chain reaction preamplification. The approach was challenged with constructed samples containing mutant and complementary DNA. The results indicated that it was possible to accurately determine SNPs with frequencies as low 0.01. The proposed approach has a great potential for realizing an accurate, sensitive, rapid, and low-cost method of SNP detection.     

Paramagnetic Particles and PNA Probe for Automated Separation and Electrochemical Detection of Influenza

Considerable efforts have been devoted to the development of rapid and sensitive methods allowing the detection of viral nucleic acid. We herein describe an assay for identification of a specific influenza sequence. The suggested method was based on isolation using paramagnetic particles coupled with electrochemical detection of isolated product. Peptide nucleic acid (PNA) was used as a probe for hybridization and identification of the influenza-derived specific sequence. The use of PNA can show numerous benefits: PNA probe is not degradable by enzymes and the duplex of PNA with RNA/DNA is more thermostable and more resistant to pH changes than DNA/DNA or RNA/RNA duplexes. This PNA probe assay can be applied as a magnetically guidable tool for detection of DNA/RNA samples under different conditions.     

Paramagnetic Particles and PNA Probe for Automated Separation and Electrochemical Detection of Influenza

Considerable efforts have been devoted to the development of rapid and sensitive methods allowing the detection of viral nucleic acid. We herein describe an assay for identification of a specific influenza sequence. The suggested method was based on isolation using paramagnetic particles coupled with electrochemical detection of isolated product. Peptide nucleic acid (PNA) was used as a probe for hybridization and identification of the influenza-derived specific sequence. The use of PNA can show numerous benefits: PNA probe is not degradable by enzymes and the duplex of PNA with RNA/DNA is more thermostable and more resistant to pH changes than DNA/DNA or RNA/RNA duplexes. This PNA probe assay can be applied as a magnetically guidable tool for detection of DNA/RNA samples under different conditions.

     

A generic sandwich-type biosensor with nanomolar detection limits

A quantitative and highly sensitive, yet simple and rapid, biosensor system was developed for the detection of nucleic acid sequences that can also be adapted to the detection of antigens. A dipstick-type biosensor with liposome amplification, based on a sandwich assay format with optical detection, was combined with a simple coupling reaction that allows the transformation of the generic biosensor components to target specific ones by a mere incubation step. This biosensor platform system was developed and optimized, and its principle was proven using DNA oligonucleotides that provided a nucleic acid biosensor for the specific detection of RNA and DNA sequences. However, the coupling reaction principle chosen can also be used for the immobilization of antibodies or receptor molecules, and therefore for the development of immunosensors and receptor-based biosensors. The generic biosensor consists of liposomes entrapping sulforhodamine B that are coated with streptavidin on the outside, and polyethersulfone membranes with anti-fluorescein antibodies immobilized in the detection zone. In order to transform the generic biosensor into a specific DNA/RNA biosensor, two oligonucleotides that are able to hybridize to the target sequence were labeled with a biotin and a fluorescein molecule, respectively. By simultaneously incubating the liposomes, both oligonucleotides, and the target sequence in a hybridization buffer for 20–30 min at 42 ^°C, a sandwich complex was formed. The mixture was applied to the polyethersulfone membrane. The complex was captured in the detection zone and quantified using a handheld reflectometer. The system was tested using RNA sequences from B. anthracis, C. parvum and E. coli. Quantitation of concentrations between 10 fmol and 1000 fmol (10–1000 nM) was possible without altering any biosensor assay conditions. In addition, no changes to hybridization conditions were required when using authentic nucleic acid sequence-based amplified RNA sequences, and the generic biosensor compared favorably with those previously developed specifically for the RNA sequences. Therefore, the universal biosensor described is an excellent tool, for use in laboratories or at test sites, for rapidly investigating and quantifying any nucleic acid sequence of interest, as well as potentially any antigen of interest that can be bound by two antibodies simultaneously.     

Effects of alkyl chain length of monomer and dye-doped type on the electro-optical properties of polymer-dispersed liquid crystal films prepared by nucleophile-initiated thiol-ene click reaction

ABSTRACT

Polymer-dispersed liquid crystal (PDLC) films containing a series of monomers with different alkyl chain lengths were prepared by nucleophile-initiated thiol-ene click reaction. The effect of alkyl chain length of monomers, dye and temperature on electro-optical properties of PDLC films was investigated. It was found that the alkyl chain length and polymerisation rate of monomers together determine the size of liquid crystal (LC) droplets, thus affecting the electro-optical properties of PDLC. In addition, the type and content of dyes could be optimised to obtain PDLC materials with better comprehensive properties for display.     

集成核酸提取的实时荧光PCR微全分析系统

集成核酸提取的实时荧光PCR微全分析系统将核酸提取、PCR扩增与实时荧光检测进行整合,在同一块微流控芯片上实现了核酸分析过程的全自动和全封闭,具有试剂用量少、分析速度快、操作简便等优点。本研究采用微机械加工技术制作集成核酸提取微流控芯片的阳极模,使用组合模具法和注塑法制作具有3D通道的PDMS基片,与玻璃基底通过等离子体键合封装成集成核酸提取芯片。构建了由微流体速度可调节(0~10 mL/min)的驱动控制装置、温控精度可达0.1℃的TEC温控平台、CCD检测功能模块等组成的微全分析系统。以人类血液裂解液为样品,采用硅胶膜进行芯片上核酸提取。系统根据设置好的时序自动执行,以2 mL/min的流体驱动速度完成20μL裂解液上样、清洗;以1 mL/min的流体驱动速度完成DNA洗脱,抽取PCR试剂与之混合注入到反应腔。提取的基因组DNA以链上内参基因GAPDH为检测对象,并以传统手工提取为对照,在该系统平台上进行PCR扩增和熔解曲线分析实验。片上PCR扩增结果显示,扩增曲线明显,Ct值分别为25.3和26.9。扩增产物进行熔解曲线分析得到的熔解温度一致,均为89.9℃。结果表明,此系统能够自动化、全封闭的在微流控芯片上完成核酸提取、PCR扩增与实时定量分析。     

Functional Monomers and Polymers Containing Nucleic Acid Bases: Experiments on Template Polymerization

In order to see how the specific base-base interactions observed between complementary nucleic acid bases can be realized for free-radical polymerization systems, the template polymerization of methacrylate type monomers containing nucleic acid bases in the presence of template polymers containing complementary bases was studied. Stereoregular polymers were chosen as the template polymers. The radical copolymerization of the monomers containing complementary nucleic acid bases was also studied in different solvents.     

Nucleic acid biosensors for environmental pollution monitoring

Nucleic acid-based biosensors are finding increasing use for the detection of environmental pollution and toxicity. A biosensor is defined as a compact analytical device incorporating a biological or biologically-derived sensing element either integrated within or intimately associated with a physicochemical transducer. A nucleic acid-based biosensor employs as the sensing element an oligonucleotide, with a known sequence of bases, or a complex structure of DNA or RNA. Nucleic acid biosensors can be used to detect DNA/RNA fragments or either biological or chemical species. In the first application, DNA/RNA is the analyte and it is detected through the hybridization reaction (this kind of biosensor is also called a genosensor). In the second application, DNA/RNA plays the role of the receptor of specific biological and/or chemical species, such as target proteins, pollutants or drugs. Recent advances in the development and applications of nucleic acid-based biosensors for environmental application are reviewed in this article with special emphasis on functional nucleic acid elements (aptamers, DNAzymes, aptazymes) and lab-on-a-chip technology.     

Sequence-specific detection of nucleic acids utilizing isothermal enrichment of G-quadruplex DNAzymes

G-quadruplex DNAzymes are peroxidase-like complexes formed by nucleic acid G-quadruplexes and hemin. Various chemical sensors and biosensors have been developed, based on such DNAzymes. Here we report a novel, specific nucleic acid detection method utilizing the isothermal amplification strategy of G-quadruplex DNAzymes. In this method, an unlabeled oligonucleotide probe was used. The probing sequence of the oligonucleotide was in the form of a stem-loop structure. A G-rich sequence, containing three GGG repeats, was linked to the 5′-end of the stem-loop structure. In the presence of target, the probing sequence hybridized to the target, and a G_n (n ≥ 2) repeat was extended from its 3′-end. This G_n repeat, together with the three GGG repeats at the 5′-end, folded into a G-quadruplex, and displayed enhanced peroxidase acitivity upon hemin binding. Utilizing the dynamic binding interaction between the probe and its target, the enrichment of G-quadruplex DNAzymes was achieved. Using this method, simple, rapid and cost-effective nucleic acid detection could be achieved. This method displayed high target-length tolerance and good detection specificity; one-base mismatch could be judged easily, even by visual inspection. This method may be used as an auxiliary tool for amplified detection of specific DNA targets in some situations, in which isothermal detection is desirable.