一种中空金/银双金属纳米颗粒的制备及其SERS性能

以银纳米颗粒为牺牲模板,利用Ag和HAu Cl4之间的置换反应,结合柠檬酸钠同步还原的方法制备了一种中空金/银双金属纳米颗粒。通过对颗粒形貌及局域表面等离子体共振(LSPR)的分析,初步研究了此类金/银纳米颗粒的生长机理,并对影响反应的因素进行了探讨。结果表明,通过控制反应条件可以实现对LSPR的精密调控。该类金/银双金属纳米颗粒可用作为SERS基底,苯硫酚在其表面增强因子可达107,并具有良好的信号重现性。该基底用于atto610标记的生物素与亲和素的SERS检测,检测限可达80 pg/m L。     

Confocal microscopy as a tool for the study of the intranuclear topography of chromosomes

A scanning confocal microscope was used to investigate the spatial positions of specific regions within blood cell nuclei. These centromeric regions were fluorescently labelled by in-situ hybridization to suspended nuclei with a centromere-1-specific DNA probe. The 3-D image data sets, obtained by optical sectioning of the cells, were used to determine the spatial position of the centromeric regions in the nuclei by means of specially developed software. The centromeres were found to be localized near the nuclear boundary. This spatial pattern was tested against a random distribution model by means of the Kolmogorov—Smirnov test. The difference between the two patterns was at a P < 0?01 significance level.     

A new method of immobilization of proteins on activated ester terminated alkanethiol monolayers towards the label free impedancemetric detection

This paper investigates a new immobilization procedure for biological molecules that is based on the formation of reactive ω-functionalized-self-assembled thiol monolayers onto a gold electrode. The homogeneous self-assembled monolayer was characterized by X-ray photoelectron spectroscopy (XPS), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The SAM modified gold electrode showed a clear peak corresponding to S_2p that characterized the Au-thiolate bond, while cyclic voltammetry and electrochemical impedance spectroscopy measurements, in 10 mM phosphate buffer pH 7, in the presence of Fe(CN)₆^{− 3/− 4} as redox probe, showed that these monolayers were densely packed and prevented electron transfer towards the gold surface. These homogeneous SAMs were used to immobilize biotin hydrazide by covalent attachment, after the nucleophilic attack of the amino group of biotin hydrazide on the ω-activated ester function of thiols. The biotin–avidin interaction was then examined as a model for an affinity biosensor with electrochemical impedance spectroscopy. A Randles equivalent circuit was used for the interpretation of impedance data and the change in the interfacial properties at the modified-electrode/electrolyte interface were monitored through charge-transfer resistance variation. The proposed affinity biosensor showed a detection range that was linear between 200 and 800 ng/ml for avidin. In order to improve the sensitivity the technique of mixed self-assembled monolayers was adopted. Mixed SAMs were elaborated by co-adsorption of two differently substituted thiols, one was substituted by a reactive group that was used to react with the amino group of biotin hydrazide, whereas the other was substituted by an hydroxyl group that was chosen to mimic protein resistance. In this study, we started with a 1:3 activated ester:hydroxyl-terminated alkanethiol ratio. The results obtained with the mixed SAMs appeared to be better than those obtained with the homogeneous SAMs, and the corresponding affinity biosensor presented two detection ranges that were linear between 20 and 100 ng/ml and between 100 and 1200 ng/ml, respectively, with two different slopes.     

Impedimetric Measurements for Monitoring Avidin-Biotin Interaction on Self-Assembled Monolayer

Avidin-biotin has been controllable immobilized on the surface of gold electrodes using mercaptopropionic acid as self-assembled monolayer. Electrochemical impedance spectroscopy (EIS) was employed to investigate the changes that appear at the electrode surface in the presence of a redox mediator, K₃[Fe(CN)₆]. An electrical model more complex than that in other studies was used to interpret the EIS measurements (Randles circuit). This model is very useful because it takes into consideration both the transfer of electrons at the electrode/electrolyte interface and the diffusion of redox species through the double layer. The model allowed us to determine some important parameters like solution resistance R_sol, charge-transfer resistance R_ct, double-layer capacitance C_dl, Warburg resistance R_W, and the diffusion time constant τ. The EIS results proved that immobilization of avidin-biotin increased the charge-transfer resistance R_ct, due to the insulating character of these molecules.     

Fabrication and Characterization of an OTFT‐Based Biosensor Using a Biotinylated F8T2 Polymer

Solution‐processable organic semiconductors have been investigated not only for flexible and large‐area electronics but also in the field of biotechnology. In this paper, we report the design and fabrication of biosensors based on completely organic thin‐film transistors (OTFTs). The active material of the OTFTs is poly(9,9‐dioctylfluorene‐co‐bithiophene) (F8T2) polymer functionalized with biotin hydrazide. The relationship between the chemoresistive change and the binding of avidin‐biotin moieties in the polymer is observed in the output and on/off characteristics of the OTFTs. The exposure of the OTFTs to avidin causes a lowering of ID at V_D = ‐40 V and V_G = ‐40 V of nearly five orders of magnitude.     

牛乳中氨苄青霉素残留的受体分析法测定

建立基于受体识别技术检测牛乳青霉素类抗生素残留的新方法。通过生物技术制备与青霉素类抗生素具有高度亲和力的受体蛋白质,进而以其为识别元件开发间接竞争氨苄青霉素残留检测方法。青霉素受体包被质量浓度5μg/mL,生物素化氨苄青霉素质量浓度500ng/mL 条件下,对其残留量进行测定,检出限为2μg/kg,有较好的精密度及回收率。在样品中残留的青霉素种类已知的前题下(以氨苄青霉素为例),本方法可以作为定量筛选方法。     

A Fluorescent “Allosteric Scorpionand” Complex Visualizes a Biological Recognition Event

We describe a new class of fluorescent reporter and its employment to visualize the biotin/avidin binding interaction. Derivatives of the azamacrocycle cyclam that contain a pendant naphthalimide dye are inherently fluorescent when zinc(II) is coordinated. Introducing a second pendant group—biotin—affords an unsymmetrical bis‐triazole‐scorpionand ligand that interacts specifically with avidin. This ligand has been assembled by using a one‐pot “double‐click” strategy and complexed with copper(II) and zinc(II). The zinc(II) complex is fluorescent, and its fluorescence output changes in the presence of avidin. Upon avidin binding, the fluorescence output is diminished by interaction with the protein, at [complex]/[avidin] ratios of up to 4:1. The observed change might arise from a specific quenching effect in the biotin binding pocket or from a binding‐induced change in the coordination geometry of the complex.     

咸鸭蛋清抗生物素蛋白的分离纯化及快速测定方法的建立

采用2-亚氨基生物素琼脂糖凝胶4B从酸热变性后咸鸭蛋清中亲和分离了抗生物素蛋白，并利用生物素化的过氧化物酶偶联起显色反应，建立了一种简便的抗生物素蛋白含量测定新方法．进而对咸鸭蛋清抗生物素蛋白进行了普查。实验结果表明：咸鸭蛋清抗生物索蛋白回收率达60．1％±5．0％，纯化倍数为222．5，纯化蛋白经SDS—PAGE电泳均显示单一蛋白染色带，其对应的分子量约为67．8K，咸鸭蛋清抗生物素蛋白质量分数约为0．05％，是抗生物索蛋白潜在的资源，本研究为咸鸭蛋清的回收利用打下了基础。     

Effect of Pre‐Filtration on Selective Isolation of Tat Protein by Affinity Membrane Separation: Analysis of Flux,Separation Efficiency,and Processing Time

Abstract

The isolation and purification of Tat protein from bacterial lysate using avidin‐biotin interaction in microfiltration membranes have been reported in the literature. To increase the efficacy of the technique, improvements in flux, Tat separation efficiency, and processing time are essential. In the current research work a pre‐filtration step was introduced to remove unwanted high molecular weight proteins and other impurities from feed prior to affinity membrane separation. Significant enhancement in flux and separation efficiency of Tat was observed. Processing time was also reduced significantly. For example, with UF pretreatment step the total Tat recovery was around four times higher (with processing time 25% lower) than that observed with the untreated feed. The quality of purified Tat was analyzed by SDS‐PAGE, Western Blot, and biotin analysis. Flux behavior in affinity separation was described by model equations.