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 Isolation of Influenza Oligonucleotide Labelled with CdS QDs

In this study, we describe hybridization design probes consisting of paramagnetic particles and quantum dots (QDs) with targeted DNA, and their application for detection of avian influenza virus (H5N1). Optical properties of QDs were beneficial, but the main attention was paid to the electroactivity of metal part of QDs and ODNs themselves. Differential pulse voltammetry was used for detection of cadmium(II) ions and square wave voltammetry for detection of cytosine–adenine peak in ODN-SH-Cd complex. It clearly follows from the obtained results that the optimized conditions were temperature of hybridization 25 °C, time of hybridization 35 min, and concentration of ODN-SH-Cd complex 20 μg mL^?1. The detection limit (3 signal/noise) was estimated as 15 ng mL^?1 of ODN-SH-Cd.     

Electrochemical Magnetoimmunosensing Approach for the Sensitive Detection of H9N2 Avian Influenza Virus Particles

A novel electrochemical magnetoimmunosensor for fast and ultrasensitive detection of H9N2 avian influenza virus particles (H9N2 AIV) was designed based on the combination of high‐efficiency immunomagnetic separation, enzyme catalytic amplification, and the biotin–streptavidin system. The reusable, homemade magneto Au electrode (M‐AuE) was designed and used for the direct sensing. Immunocomplex‐coated magnetic beads (IMBs) were easily accumulated on the surface of the M‐AuE to obtain the catalytically reduced electrochemical signal of H₂O₂ after the immunoreaction. The transducer was regenerated through a simple washing procedure, which made it possible to detect all the samples on a single electrode with higher reproducibility. The magnetic‐bead‐based electrochemical immunosensor showed better analytical performance than the planar‐electrode‐based immunosensor with the same sandwich construction. Amounts as low as 10 pg mL^?1 H9N2 AIV could be detected even in samples of chicken dung. This electrochemical magnetoimmunosensor not only provides a simple platform for the detection of the virus with high sensitivity, selectivity, and reproducibility but also shows great potential in the early diagnosis of diseases.     

Photo- and Electrochemical Dual-Responsive Iridium Probe for Saccharide Detection

Dual detection systems are of interest for rapid, accurate data collection in sensing systems and in vitro testing. We introduce an Ir^III complex with a boronic acid receptor site attached to the 2-phenylpyridine ligand as an ideal probe with photo- and electrochemical signals that is sensitive to monosaccharide binding in aqueous solution. The complex displays orange luminescence at 618 nm, which is reduced by 70 and 40 % upon binding of fructose and glucose, respectively. The electro-chemiluminescent signal of the complex also shows a direct response to monosaccharide binding. The Ir^III complex shows the same response upon incorporation into hydrogel matrices as in solution, thus demonstrating the potential of its integration into a device, as a nontoxic, simple-to-use tool to observe sugar binding over physiologically relevant pH ranges and saccharide concentrations. Moreover, the complex's luminescence is responsive to monosaccharide presence in cancer cells.     

Electrochemical Detection of Avian Influenza Virus Genotype Using Amino‐ssDNA Probe Modified Gold Electrodes

A DNA biosensor for the detection of specific oligonucleotide sequences of Avian Influenza Virus type H5N1 has been proposed. The NH₂‐ssDNA probe was deposited onto a gold electrode surface to form an amide bond between the carboxyl group of thioacid and the amino group from ssDNA probe. The signals generated as a result of hybridization were registered in square wave voltammetry and electrochemical impedance spectroscopy in the presence of [Fe(CN)₆]^3?/4? as a redox marker. The genosensor is capable to determine 20‐mer and 180‐bp (PCR products) oligonucleotides complementary sequences with detection limit in the fM range. The genosensor displays good selectivity and sensitivity. The 20‐mer as well as 180‐bp oligonucleotides without a complementary sequence generate very low signal.     

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.     

PNA探针与DNA探针的系统比较

肽核酸（Peptide Nucleic Acid,PNA）是近十几年发展起来的以中性酰胺键为骨架的脱氧核糖核酸（Deoxyribonucleic Acid,DNA）类似物,其结构介于多肽和DNA之间。由于PNA能够与DNA和RNA特异性地结合,可以制备PNA探针。与DNA探针相比,其杂交的稳定性和特异性增加且能在低盐浓度下进行杂交。本文从DNA和PNA的分子结构和性质、DNA探针和PNA探针的设计制备、杂交亲和性、杂交动力学以及在生物传感器上的应用等方面进行了系统比较。     

Magnetic Hetero-flocculation of Paramagnetic Colloidal Particles

The feasibility of a high-gradient magnetic separation process, utilizing magnetite as the energizable element in lieu of stainless steel wool, is evaluated by means of an equilibrium, two-particle, magnetic hetero-flocculation model. The model calculates the net force, defined as the sum of the magnetic, electrostatic, and van der Waals forces, exerted on a paramagnetic nanoparticle that is in the proximity of a fixed magnetite particle. Since the nanoparticle-magnetite system is assumed to be in direct contact with the moving fluid, the influence of the hydrodynamic force on the magnetic attractive force between the two particles is also explored. This model clearly reveals the ranges and conditions over which each of these various forces contributes to the net force relative to Brownian (thermal) motion. The model also reveals the feasibility of using magnetite particles instead of stainless steel as the energizable element for high-gradient magnetic separation. Important variables investigated include the size and surface charge of the particles, the magnetic field, the flow velocity, the electrolyte concentration, and the magnetic susceptibility of the nanoparticle. Copyright 2000 Academic Press.     

Ferrocene-derivative Electrochemical Probe for the Selective Detection of Carcinoma-associated STn Antigen

Ferrocene-derivatives have a well-defined electrochemical behaviour, which can be changed upon binding to other molecules (including nonelectroactive species), as the conjugate presents a slower diffusion to the electrode surface. Therefore, the binding event can be easily detected by monitoring the decrease in the current intensity of the ferrocene-derivative oxidation peak. An electrochemical probe composed of ferrocenecarboxylic acid conjugated with Sambucus nigra agglutinin was prepared and used to selectively detect the STn antigen, a pan-carcinoma glycobiomarker. The probe showed high selectivity towards the STn antigen present in glycoproteins and demonstrated effectiveness in the analysis of serum samples from carcinoma patients.     

Discriminative Detection of Biothiols by Electron Paramagnetic Resonance Spectroscopy using a Methanethiosulfonate Trityl Probe

Biothiols, such as glutathione (GSH), homocysteine (Hcy), and cysteine (Cys), coexist in biological systems with diverse biological roles. Thus, analytical techniques that can detect, quantify, and distinguish between multiple biothiols are desirable but challenging. Herein, we demonstrate the simultaneous detection and quantitation of multiple biothiols, including up to three different biothiols in a single sample, using electron paramagnetic resonance (EPR) spectroscopy and a trityl‐radical‐based probe (MTST). We term this technique EPR thiol‐trapping. MTST could trap thiols through its methanethiosulfonate group to form the corresponding disulfide conjugate with an EPR spectrum characteristic of the trapped thiol. MTST was used to investigate effects of l ‐buthionine sulfoximine (BSO) and pyrrolidine dithiocarbamate (PDTC) on the efflux of GSH and Cys from HepG2 cells.     

基于双氢青蒿素-巯基化合物电化学探针对巯基化合物的检测

在20%乙醇+B-R缓冲溶液(pH7.2)介质中,利用双氢青蒿素与谷胱甘肽、6-巯基嘌呤及L-半胱氨酸作用,分别于-1.05,-1.20和-1.10V处获得双氢青蒿素-巯基化合物复合物灵敏的还原峰,该还原峰在一定浓度范围内随着巯基化合物以及双氢青蒿素浓度的增加而增大,因此可将其作为电化学探针应用于上述3种巯基化合物以及双氢青蒿素的检测,为检测巯基化合物提供了一种新方法;探讨了复合物形成的电极反应机理,利用纳米金与巯基的共价作用阻断巯基参与形成复合物,证实了复合物是由双氢青蒿素中的碳自由基与巯基化合物巯基中的硫原子通过SC键结合形成。     

The Separation and Determination of Molybdenum by Electron Paramagnetic Resonance

Abstract

A procedure is described for the separation and quantitative determination of molybdenum by means of EPR. Molybdenum(VI) was precipitated from a hydrochloric acid solution (1.5 Molar) with α-benzoinoxime. The precipitate was separated by centrifugation and then the sample was dried in an oven at 105°C. Nitrogen was passed over the sample to complete the drying procedure. The precipitate was dissolved in acetonitrile which was 0.5 M in lithium perchlorate. Electrochemical reduction of the sample produced the molybdenum(V) species. The concentration of molybdenum(V) was determined directly from the intensity of the first derivative EPR signal. The range of linearity of the analytical curve was 1.00 × 10^?2 M to 1.00 × 10^?4 M, and a routine accuracy of less than 6% was obtained.     

基于DNA四面体纳米结构探针的电化学生物传感器检测DNA甲基化

该文以DNA四面体纳米结构探针(TSP)为捕获探针,将辣根过氧化物酶标记的IgG抗体结合在纳米金颗粒表面(AuNPs-IgG-HRP)作为信号分子,构建了一种新型DNA甲基化电化学传感器。利用一步热变性法组装成TSP后,通过Au—S键固定在修饰纳米金颗粒的金电极表面,经过靶标DNA杂交、5-甲基胞嘧啶(5-mc)抗体及AuNPs-IgG-HRP结合后,用差分脉冲伏安法(DPV)进行检测。采用循环伏安法(CV)和电化学阻抗谱(EIS)对修饰电极的构建过程进行电化学表征。探究了杂交时间、5-mc抗体浓度、IgG-HRP加入体积、氢醌(HQ)和过氧化氢(H₂O₂)浓度对传感器的影响。在最佳条件下,该传感器对甲基化DNA的线性响应范围为1.0×10^-15～1.0×10^-10 mol/L,检出限(S/N=3)为4.4×10^-16 mol/L。该传感器具有良好的选择性和稳定性,为DNA甲基化检测提供了新方法。     

一种新的测定微粒的电化学技术

设计了一种由2个石墨电极短路相连组成工作电极的新的电化学池装置.操作时首先通过外力按压使极少量固体微粒粘附在其中一个石墨电极表面上,然后在溶液存在下将微粒夹紧并固定在2个石墨电极表面之间进行电化学测定.电化学转化过程中生成的可溶性物质被封闭在2个石墨电极表面之间而得到测定.用该技术对钯沉积在氧化铝上而组成的催化剂的电化学行为以及黄铁矿的电化学行为进行了研究.结果表明,其兼具可电解粘合剂碳糊电极和固体微粒伏安法(voltammetry of microparticles)技术的优点而避免了各自的缺点:即不使用粘合剂,从而消除了粘合剂中杂质产生的氧化或还原电流的影响;可测定电化学转化过程中生成的可溶性物质;分辨率好、易于操作.     

ZnO超微粒子的EPR特性和光催化性能

利用XRD,TEM,XPS和EPR等研究了ZnO超微粒子的EPR特性和光催化性能.前驱物碱式碳酸锌在320,430,550和700℃经热处理制得的ZnO超微粒子粒径分别为13.5,19.3,26.1和38.5nm,属六方晶系纤锌矿结构;室温下ZnO超微粒子表现出稳定的单一谱线的EPR信号,其强度随粒径的增大而减小.而在液氮温度下,ZnO超微粒子的EPR谱具有6条强度不等的超精细谱线,在光催化氧化C₇H₁₆和SO₂过程中,其光催化活性随其EPR信号强度的减小而下降.说明O₂-空位在光催化反应中起重要作用.     

基于锁核酸探针的传感器用于BCR/ABL融合基因检测的研究

基于慢性粒细胞白血病中BCR/ABL融合基因的碱基序列,设计了一种新型发夹结构锁核酸(locked nucleic acids, LNA)探针,把LNA探针通过Au-s键固定在金电极表面构建了特异的生物传感器.LNA探针与目标链DNA杂交,以自行合成的苯甲酸二聚铜配合物([Cu2(C7H5O2)4(C2H6O)2], 简称[Cu(R)2]2+)为杂交指示剂,应用差示脉冲伏安法进行检测,表现出良好的响应信号.该新型锁核酸传感器能较好的区分完全互补链DNA、单碱基错配链DNA.对互补链DNA检测的线性范围为1.0×10-8～1.0×10-6 mol•L-1,检出限为2.0×10-9 mol•L-1.     

Scanning Electrochemical Microscopy for Electrochemical Detection of Single‐base Mismatches by Tagging Ferrocenecarboxylic Acid as a Redox Probe to DNA

Scanning electrochemical microscopy (SECM) was employed for sensitive detection of single base mismatches (SBMs) in a sandwiched dsDNA. Ferrocenecarboxylic acid (Fc), covalently conjugated to the dsDNA, was oxidized to Fc⁺ via the DNA‐mediated charge transfer from the underlying gold substrate, and reduced back to Fc by SECM tip generated ferrocyanide. The electrocatalytic oxidation of SECM tip‐generated ferrocyanide was sensitive to presence, as well as the type of SBMs. Apparent standard rate constants (k⁰_app) values for different SBMs, both near the electrode surface and far from it, were evaluated by SECM. The method can detect SBMs independent of their position in dsDNA.     

微流控芯片电化学与电化学发光检测的某些进展

近年来,微全分析系统(μTAS)发展很快^[1~3],已有多种检测器被应用.电化学检测法具有微型和原位的特点,可为微环境提供高灵敏度的检测效果.     

Electrochemical Detection of Cecropin CM4 Gene by Single Stranded Probe and Cysteine Modified Gold Electrode

ABSTRACT

A single stranded Cecropin CM4 gene (108 bases) was further immobilized at a cysteine modified gold electrode with the help of water soluble 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC). The ssDNA at the modified electrode could undergo hybridization in a hybridization buffer containing single stranded plasmid pLCM-4SN. A DNA minor groove binder, Hoechst 33258, was employed to discriminate between ssDNA and dsDNA. The anodic waves in differential pulse voltammograms (DPVs), of Hoechst 33258 bound to the DNAs, were used as the indicator. This assay procedure was shown to be rapid, sensitive and precise, thus a kind of prototype DNA biosensor was developed.     

微囊藻属DNA电化学传感器的研制

利用自组装法将巯基修饰的DNA探针与6-巯基-1-己醇(MCH)固定到金电极表面,制备了微囊藻属特定DNA传感器,将该传感器与完全互补的微囊藻DNA序列、完全不互补序列,以及单碱基错配序列进行杂交,以Hoechst 33258为杂交指示剂,应用循环伏安法和线性扫描伏安法研究了该传感器对目标DNA的电化学检测行为.研究表明,当与完全互补DNA杂交后,Hoechst 33258氧化信号有明显的增强.实验对自组装时间、MCH浸泡时间及杂交液离子浓度进行了优化.结果表明,当自组装时间为90 min,MCH浸泡时间为1 h,杂交溶液中NaCl浓度为0.3 mol/L时,电化学信号最好.目标DNA的氧化峰电流值与其浓度在1×10~(-8) ～1×10~(-6) mol/L范围内呈良好的线性关系,检出限为8.1×10~(-9) mol/L.