金标记羟胺放大化学发光检测赭曲霉毒素A

以羧基磁性微球为分离载体,连接氨基捕获探针和适配体,加入生物素化报告序列和赭曲霉毒素A(Ochratoxin A,OTA)竞争结合适体,继续加入链霉亲和素纳米金和羟胺/Au~(3+)以显著提高化学发光检测OTA的灵敏度,从而建立了一种纳米金标记羟胺放大化学发光检测OTA的高灵敏度方法。优化了羧基磁性微球、氨基捕获探针、适配体、生物素化报告序列、链霉亲和素纳米金的用量。优化条件下,在OTA质量浓度0.01~50 ng/m L范围内,化学发光信号值与OTA浓度的对数呈较好的线性关系(r~2=0.992 5),检出限为1.58×10~(-3)ng/mL。对啤酒样品进行OTA加标回收实验,回收率为97.4%~105.4%,相对标准偏差为4.0%~5.5%。     

基于磁性纳米颗粒和金纳米粒子构建DNA电化学生物传感技术

本文构建了一种基于纳米粒子、茎环DNA和丝网印刷电极(SPCE)的电化学生物传感技术用于乳腺癌基因的快速、灵敏检测。该传感技术中,探针DNA的两端分别标记了巯基和生物素,巯基用于与金纳米粒子(AuNPs)作用,生物素用于与磁性纳米颗粒(MNPs)表面修饰的链酶亲和素作用以达到富集的目的,之后利用SPCE进行电化学检测。无目标DNA存在时,双标记DNA保持茎环结构,使得生物素分子很难和MNPs上的亲和素接触。一旦加入目标DNA,茎环结构打开,生物素得以与MNPs上的链霉亲和素发生特异性结合,形成的复合物(MNPs-DNA-AuNPs)通过磁性富集到SPCE表面,从而获得AuNPs的电化学信号。该DNA电化学生物传感对单碱基错配有良好的分辨能力,完全互补DNA的检出限为8.0×10-13 mol/L。     

基于磁性辅助的杂交链反应放大检测三磷酸腺苷

本文提出了一种基于磁性辅助的杂交链反应放大检测三磷酸腺苷(ATP)的传感策略。磁性纳米粒子表面易于修饰,而且操作方便,具有很好的分离效果,能够提高生物传感的选择性。首先,利用生物素与链霉亲和素之间的亲和力作用,将生物素标记的ATP核酸适配体连接到链霉亲和素修饰的磁性纳米粒子表面,加入与ATP核酸适配体互补的一段DNA进行杂交,通过磁性分离除去未杂交上的DNA,加入靶向ATP,ATP与其适配体特异性结合将适配体的互补链通过磁性分离出来,磁性分离出的信号DNA继续用于下一步的杂交链反应,将信号放大,最后利用氧化石墨烯(GO)对荧光的猝灭效应降低背景荧光,达到高灵敏度、高选择性检测靶向ATP。其中,ATP的最低检测浓度为0.1nmol/L。     

应用纳米磁性球电化学检测特定序列DNA

采用分散聚合法制备纳米磁性羧基球,利用化学偶联法将末端修饰氨基的寡聚核苷酸固定在纳米磁性球表面,制成新型核酸探针,该探针可特异性结合目标单链寡聚核苷酸.在磁场作用下,将纳米磁珠与本体溶液分离并富集在电极表面,以中性红为嵌合指示剂,用示差脉冲伏安法测定杂交结果.经过条件优化,本法测定DNA的浓度线性范围为1.0×10^-6～5.0×10^-9mol/L,检出限为8.6×10^-10mol/L.     

基于磁性微球的无标记化学发光端粒传感新技术

近年来无标记型DNA传感技术的研究已成为病原基因测定和基因疾病诊断等领域新的研究热点之一. 基于磁性微球分离和富集的方法, 建立了一种新型的无标记化学发光检测技术, 并成功地应用于特定序列DNA——端粒的检测. 首先采用dT₂₀修饰的磁性微球, 与连接有dA₂₀的捕获探针DNA杂交, 然后再与端粒进行第二步杂交反应. 磁性分离洗涤后, 利用端粒中富含的G碱基与3,4,5-三甲氧基苯乙二醛反应产生特异性化学发光, 从而实现特定序列 DNA——端粒的无标记检测. 实验结果表明: 该法具有操作简便、分析快速、灵敏度高、专属性好等特点. 目标DNA浓度在5×10^－9～1×10^－7 mol/L浓度范围内具有良好的线性关系, 相关系数为0.9918.     

磁性核壳介孔氧化硅微球的制备与应用

磁性核壳介孔氧化硅微球作为一种新型功能复合材料,已成为众多研究领域的一个研究热点。本文综述了近年来利用模板法合成磁性核壳介孔氧化硅微球的研究进展,重点阐述了溶胶-凝胶法和微乳液法在实心微球和中空微球制备中的应用。介绍了磁性介孔二氧化硅微球在蛋白质、DNA分离,靶向药物传输等生物医学上的应用以及磁性酸催化、加氢催化、纳米贵金属催化、光催化等催化领域的应用,并对其未来的发展趋势做了展望。     

基于纳米复合材料探针的新型棉线DNA室温快速检测装置的研究

建立了一种简单灵敏的棉线快速可视化DNA分析方法.采用碳纳米管/金纳米粒子复合材料修饰发夹型结构DNA探针构建信号探针,DNA探针两端各有8个A碱基,中间序列则与目标DNA链完全匹配,3'端修饰生物素,5'端修饰巯基,在甲氧襞因存在下,DNA探针因为两端的A碱基与甲氧襞因相互作用而形成发夹型结构.当样品中存在目标DNA...     

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.     

荧光磁性双功能树状分子微球的制备与表征

采用化学共沉淀法, 以FeCl₃·6H₂O和FeSO₄·7H₂O为原料制备了磁性Fe₃O₄纳米颗粒, 采用树状大分子对其进行修饰, 然后通过树状大分子具有的大量空腔及末端丰富的氨基, 经吸附、 键合, 与大量巯基乙酸修饰的CdSe/CdS量子点连接, 得到三代具有荧光磁性双功能的树状分子微球, 并对其进行结构表征与性能测试. 结果表明: 三代复合后的微球的平均粒径分别为15, 34和49 nm; 一代荧光磁性微球的发光性能最佳, 其量子产率达24.1%; 零代荧光磁性微球磁性能最优, 其饱和磁化强度为15.96 A·m²/kg. 这种具有荧光和磁性的双功能纳米复合微粒有望在免疫检测、 靶向治疗、 荧光追踪和磁性分离等方面得到广泛应用.     

功能化纳米金增强的DNA电化学检测和序列分析

用冠以大量二茂铁的纳米金微粒 /抗生蛋白链菌素结合物为标记物 ,将其标记于生物素修饰的寡聚核苷酸片段上 ,制成了具有电化学活性和纳米金放大作用的DNA电化学生物传感器 .首先采用巯基DNA和巯基烷烃混合自组装膜制备了金修饰电极 ,将探针DNA分子固定在了电极表面 ,运用杂交原则结合靶点分子在电极表面形成了双螺旋的DNA链 ,然后借助抗生蛋白链菌素和生物素之间的强亲和作用 ,引入了功能化的纳米金 .通过伏安法测定了修饰在纳米金上的二茂铁的氧化还原电流 ,可以识别和测定溶液中互补的靶点DNA ,17 mer靶点DNA的浓度在 0 .0 0 1～ 10nmol/L范围内有线性关系 ,检测限可达 0 .75× 10 -12 mol/L .     

Streptavidin-modified magnetic poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) microspheres for selective isolation of bacterial DNA

The dispersion copolymerization of 2-hydroxyethyl methacrylate (HEMA) with glycidyl methacrylate (GMA) in a toluene/2-methylpropan-1-ol mixture in the presence of Fe₃O₄ nanoparticles coated with oleic acid, produced monodisperse magnetic poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) (P(HEMA-GMA)) microspheres. Oxirane groups of the microspheres were ammonolyzed and then functionalized with streptavidin using cyanuric chloride. The final product contained 0.67 mg of streptavidin per gram of wet magnetic P(HEMA-GMA) microspheres. The microspheres were characterized by elemental analysis, scanning electron microscopy, IR, UV–VIS and atomic absorption spectroscopy. The streptavidin-modified magnetic P(HEMA-GMA) microspheres were used for immobilization of biotinylated DNA and subsequent selective isolation of target DNA from complex samples using DNA/DNA hybridisation. Based on the highly selective recognition of streptavidin with a biotin-labeled DNA probe, DNA sensor was constructed for magnetic separation of DNA from real samples.     

基于磁性分离-硫化铜纳米粒子标记的DNA流动注射化学发光检测

本文基于磁性粒子(MB)良好的分离、富集能力,研究了硫化铜纳米粒子标记的流动注射-化学发光(FI-CL)DNA检测体系.通过硫化铜标记的探针1与目标DNA及连有磁球的探针2形成三明治结构,实现对目标DNA的捕获、分离与标记;通过其溶解释放出CuS标记颗粒的铜离子,引起化学发光信号增强,实现了目标DNA序列的定性定量检测.该方法对完全互补单链DNA(ssDNA)检测的线性范围为1.0×10-11～1.6×10-9 mol/L,检出限为3.0×10-12 mol/L,对1.0×10-9 mol/L目标DNA测定的相对标准偏差为3.2%(n=11),对目标碱基序列具有良好的识别能力.     

Amplified Electrochemical DNA Sensor Based on Polyaniline Film and Gold Nanoparticles

In this work, an electrochemical DNA biosensor, based on a dual signal amplified strategy by employing a polyaniline film and gold nanoparticles as a sensor platform and enzyme‐linked as a label, for sensitive detection is presented. Firstly, polyaniline film and gold nanoparticles were progressively grown on graphite screen‐printed electrode surface via electropolymerization and electrochemical deposition, respectively. The sensor was characterized by scanning electron microscopy (SEM), cyclic voltammetry and impedance measurements. The polyaniline‐gold nanocomposite modified electrodes were firstly modified with a mixed monolayer of a 17‐mer thiol‐tethered DNA probe and a spacer thiol, 6‐mercapto‐1‐hexanol (MCH). An enzyme‐amplified detection scheme, based on the coupling of a streptavidin‐alkaline phosphatase conjugate and biotinylated target sequences was then applied. The enzyme catalyzed the hydrolysis of the electroinactive α‐naphthyl phosphate to α‐naphthol; this product is electroactive and has been detected by means of differential pulse voltammetry. In this way, the sensor coupled the unique electrical properties of polyaniline and gold nanoparticles (high surface area, fast heterogeneous electron transfer, chemical stability, and ease of miniaturisation) and enzymatic amplification. A linear response was obtained over a concentration range (0.2–10 nM). A detection limit of 0.1 nM was achieved.     

Magnetic porous carbon nanocomposites derived from metal-organic frameworks as a sensing platform for DNA fluorescent detection

Metal-organic frameworks (MOFs) have emerged as very fascinating functional materials due to their tunable nature and diverse applications. In this work, we prepared a magnetic porous carbon (MPC) nanocomposite by employing iron-containing MOFs (MIL-88A) as precursors through a one-pot thermolysis method. It was found that the MPC can absorb selectively single-stranded DNA (ssDNA) probe to form MPC/ssDNA complex and subsequently quench the labelled fluorescent dye of the ssDNA probe, which is resulted from the synergetic effect of magnetic nanoparticles and carbon matrix. Upon the addition of complementary target DNA, however, the absorbed ssDNA probe could be released from MPC surface by forming double-stranded DNA with target DNA, and accompanied by the recovery of the fluorescence of ssDNA probe. Based on these findings, a sensing platform with low background signal for DNA fluorescent detection was developed. The proposed sensing platform exhibits high sensitivity with detection limit of 1 nM and excellent selectivity to specific target DNA, even single-base mismatched nucleotide can be distinguished. We envision that the presented study would provide a new perspective on the potential applications of MOF-derived nanocomposites in biomedical fields.     

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.     

超顺磁性DNA纳米富集器应用于痕量寡聚核苷酸的富集

随着纳米技术的迅速发展 ,纳米材料逐渐被应用到细胞生物学和分子生物学研究领域 ,为生物医学的研究和发展提供了新的技术和手段 [1～ 4 ] .如超顺磁性纳米颗粒由于具有尺寸小、比表面积大、悬浮稳定性好及在外磁场作用下的磁导向性运输和富集等优良特性 ,使其在细胞和生物活性     

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.     

An efficient method for recovery of target ssDNA based on amino-modified silica-coated magnetic nanoparticles

In this paper, an improved recovery method for target ssDNA using amino-modified silica-coated magnetic nanoparticles (ASMNPs) is reported. This method takes advantages of the amino-modified silica-coated magnetic nanoparticles prepared using water-in-oil microemulsion technique, which employs amino-modified silica as the shell and iron oxide as the core of the magnetic nanoparticles. The nanoparticles have a silica surface with amino groups and can be conjugated with any desired bio-molecules through many existing amino group chemistry. In this research, a linear DNA probe was immobilized onto nanoparticles through streptavidin conjugation using covalent bonds. A target ssDNA(I) (5′-TMR-CGCATAGGGCCTCGTGATAC-3′) has been successfully recovered from a crude sample under a magnet field through their special recognition and hybridization. A designed ssDNA fragment of severe acute respiratory syndrome (SARS) virus at a much lower concentration than the target ssDNA(I) was also recovered with high efficiency and good selectivity.     

An electrochemical DNA sensor based on a layers-film construction modified electrode

This work developed a relatively inexpensive and layers-film construction electrochemical sensor for DNA recognition and its performance was investigated. The Fe(3)O(4) magnetic nanoparticles-cysteine were immobilized on the carbon paste electrode (CPE) surface using magnetic force. Multiwalled carbon nanotubes (MWCNTs), gold nanoparticles (GNPs), and chitosan (Chi) were used successively to coat on the electrode surface. The thiolated capture probe was assembled and competitively hybridized with the target nucleic acid and biotinylated response probe. The electrochemical behavior was analyzed by cyclic voltammetry and electrochemical impedance spectroscopy. In addition, the sensor performance was also analyzed by introducing the notion of detection efficiency. The experimental results showed that although the electron transfer capability of the CPE is less strong than that of a metal electrode used in the DNA sensor, the materials modified on the CPE could significantly improve the performance. A detection limit of 1 nM of target DNA and a sensitivity of 2.707 × 10(3) mA M(-1) cm(-2) were obtained. Although the resulting detection limit was not remarkable, further experiments could improve it.     

Development of a photodiode array biochip using a bipolar semiconductor and its application to detection of human papilloma virus

We describe a DNA microarray system using a bipolar integrated circuit photodiode array (PDA) chip as a new platform for DNA analysis. The PDA chip comprises an 8 × 6 array of photodiodes each with a diameter of 600 μm. Each photodiode element acts both as a support for an immobilizing probe DNA and as a two-dimensional photodetector. The usefulness of the PDA microarray platform is demonstrated by the detection of high-risk subtypes of human papilloma virus (HPV). The polymerase chain reaction (PCR)-amplified biotinylated HPV target DNA was hybridized with the immobilized probe DNA on the photodiode surface, and the chip was incubated in an anti-biotin antibody-conjugated gold nanoparticle solution. The silver enhancement by the gold nanoparticles bound to the biotin of the HPV target DNA precipitates silver metal particles at the chip surfaces, which block light irradiated from above. The resulting drop in output voltage depends on the amount of target DNA present in the sample solution, which allows the specific detection and the quantitative analysis of the complementary target DNA. The PDA chip showed high relative signal ratios of HPV probe DNA hybridized with complementary target DNA, indicating an excellent capability in discriminating HPV subtypes. The detection limit for the HPV target DNA analysis improved from 1.2 nM to 30 pM by changing the silver development time from 5 to 10 min. Moreover, the enhanced silver development promoted by the gold nanoparticles could be applied to a broader range of target DNA concentration by controlling the silver development time. Figure An optical image of the PDA chip and target DNA detection through silver enhancement Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.