An optical surface plasmon resonance biosensor for determination of tetanus toxin

Surface plasmon resonance (SPR) has been successfully applied for the simple, rapid, and label-free assay of various biomolecules. This assay evaluates a novel wavelength modulation SPR biosensor for the detection of tetanus toxin. The wavelength modulation SPR biosensor is designed based on fixing the incident angle of light and measuring the reflected intensities in the resonance wavelength range spanning 400-800 nm simultaneously. Tetanus toxin (TeNT), one of the most potent toxins known, is synthesized as a 150 kDa single polypeptide chain. The SPR biosensor has been shown to be capable of directly detecting concentration of tetanus toxin as low as 0.028 Lf ml⁻¹. Under selected experimental conditions, the SPR biosensor has a good reproducibility, sensitivity and reversibility. The results illustrate how wavelength modulation SPR biosensor can be used to detect biomolecular interactions.     

Saccharide-branched Cyclodextrins as Targeting Drug Carriers

A synthetic series of heptakis-galactose-branched cyclodextrins (termed CDs) having a longer spacer arm using two amino-caproic acids as an enlarging unit were prepared. Starting with heptakis-amino-β-CD or heptakis-amino-caproic-amide-β-CD, treated with galactosyl-glucono-amide-caproic acid, the new compounds heptakis (Gal-cap1)-CD (4) or heptakis (Gal-cap2)-CD (5) were obtained. The longer galactose spacer arm extremely favors the PNA association. The effect of branch length on K with PNA was enhanced up to 138-fold 3 as well as with DXR enhanced up to 81-fold. Hexakis (Gal-cap2)-CD (6) was prepared and the association constants with rat liver cells were observed to be 2.5 × 10¹⁰ M⁻¹. A multi-high mannose type oligosaccharide branched CD (7) showed a large association constant with DXR up to 1.1 × 10⁹ M⁻¹. The two-dimensional map for the association constants of newly synthesized oligosaccharide-branched CDs toward lectin or liver cells versus the association constants toward a drug (doxorubicin) suggested a method of finding a better targeting drug carrier. The structural effect of the oligosaccharide-CDs showed that the number and length of the branch were dominant factors in designing for enhanced dual recognition.     

表面等离子共振传感器的原理与进展

表面等离子共振光谱(Surface Plasmon Resonance,SPR)是近年来得到快速发展的一门技术。它是一种无标记的、可用于实时定量检测某些固定于传感芯片上的组分与被结合物种间的绑定亲合度(binding affinity)、且可用于对相对小量物质进行检测的重要手段。由于它可方便地研究不同生物或化学物种的有关反应与动力学问题,因此具有重要的实用意义,受到广泛关注和重视。本文对有关等离子共振现象的形成及其作为敏感检测手段的机制、原理和改进等问题作了简要的介绍。     

Sequential-dissociation kinetics of non-covalent complexes of DNA with multiple proteins in separation-based approach: General theory and its application

Binding of multiple proteins to DNA is crucial in many regulatory cellular processes. The kinetics of assembly and disassembly of DNA–multiple protein complexes is very difficult to study in detail due to the lack of suitable experimental approaches. A separation-based approach has been recently proposed to resolve disassembly kinetics of such complexes. While conceptually simple, the separation-based approach generates experimental data with very complex patterns. The analysis of these patterns is a challenging problem on its own. Here we report on a mathematical approach that can extract a solution for the experimental data obtained in separation-based analysis of sequential dissociation of a DNA complex with multiple proteins. This case describes the dissociation of proteins one-by-one from the complex. Generally speaking, a mathematical solution of such problems requires calculations of multiple integrals. Our approach reduces this procedure to taking double integrals and constructing their superposition. We tested this approach with the experimental data obtained for three-step sequential dissociation of complexes of DNA with two protein copies.     

纳米金标记抗体增强SPR检测大肠杆菌O157∶H7

将金纳米粒子(AuNPs)标记的大肠杆菌O157∶H7(E.coli O157∶H7)的多克隆抗体(PAb)作为二抗,采用氨基偶联法将PAb固定在传感器表面作为一抗,通过三明治方法用双通道表面等离子体子共振(SPR)传感器对E.coli O157∶H7进行检测,并与SPR直接法检测进行了比较.结果表明,直接法的检出限为103cfu/mL,线性范围为103～109cfu/mL;AuNPs增强三明治法的检出限为10 cfu/mL,线性范围为10～1010cfu/mL,灵敏度比直接法提高了100倍,且具有更宽的检测范围.本方法不仅检测时间短,而且具有良好的选择性和重现性.     

Controllable Release and High‐Efficiency Collection of Hydrogen Peroxide: Application on the Quantitative Investigation of Biomolecule Oxidation Induced by Reactive Oxygen Species

Hydrogen peroxide and hydroxyl radical, both important members of the reactive oxygen species (ROS) family, can cause serious oxidative damages in biological systems. In order to proclaim and prevent oxidation stress, researches on the biomolecule oxidation induced by H₂O₂ or OH^. are in crucial need. However, due to the high reactivity of ROS, traditional methods are difficult to achieve the in situ quantitative investigations on those reactions involving ROS. In this work, using scanning electrochemical microscopy technique (SECM) in a tip generation‐substrate collection mode (TG‐SC), the controllable release and the high‐efficiency collection of electrogenerated H₂O₂ were achieved. Compared to ex situ fluorescence method, SECM improved the collection efficiency approximately two times larger. Based on it, SECM combined with surface plasmon resonance (SPR) was employed to in situ monitor the protein oxidation (taking Cu₁₂⁺? MT as a model) induced by H₂O₂. OH^., which was generated from the interaction between H₂O₂ and Cu₁₂⁺? MT, can attack the peptide chain and induced the unrepairable protein oxidation damage. The whole process was quantitatively characterized by SPR, and the linear relationship between SPR dip shift and the amounts of released H₂O₂ was successfully built. Our work proves that the combined SECM‐SPR technique can realize the in situ quantitative determinations of the biomolecule oxidation induced by ROS, which affords an avenue for further elucidation on the mechanisms of oxidation stress in organisms.     

基于SPR光谱分析的液体折射率测量研究

文章从电磁场理论和射线理论的角度,详细讨论了光纤表面等离子体波传感器工作原理,分析了表面等离子体共振(SPR)光谱共振波长与液体介质折射率之间的对应关系。采用相关光谱检测技术,获得八种不同类型分析醇所对应的SPR光谱曲线族,其共振波长随液体折射率的增大而逐渐向长波长方向偏移。通过对乙醇与乙二醇混合液体所对应的共振光谱分析,实现对两者配比浓度的测定。整个传感系统结构简单,测量光路部分实现全光纤化,不仅能够对目标测量介质的实时、快速、精确测量,还可用于特殊测量场合,实现远程遥测功能。     

Discovery of Novel Pleuromutilin Derivatives as Potent Antibacterial Agents for the Treatment of MRSA Infection

A series of novel pleuromutilin derivatives containing nitrogen groups on the side chain of C14 were synthesized under mild conditions. Most of the synthesized derivatives displayed potent antibacterial activities. Compound 9 was found to be the most active antibacterial derivative against MRSA (MIC = 0.06 μg/mL). Furthermore, the result of time-kill curves showed that compound 9 had a certain inhibitory effect against MRSA in vitro. Moreover, according to a surface plasmon resonance (SPR) study, compound 9 (K_D = 1.77 × 10⁻⁸ M) showed stronger affinity to the 50S ribosome than tiamulin (K_D = 2.50 × 10⁻⁸ M). The antibacterial activity of compound 9 was further evaluated in an MRSA-infected murine thigh model. Compared to the negative control group, tiamulin reduced MRSA load (~0.7 log₁₀ CFU/mL), and compound 9 performed a treatment effect (~1.3 log₁₀ CFU/mL). In addition, compound 9 was evaluated in CYP450 inhibition assay and showed only moderate in vitro CYP3A4 inhibition (IC₅₀ = 2.92 μg/mL).     

SPR免疫竞争法研究小分子吗啡与其抗体间作用的动力学特征

表面等离子体共振技术(SPR)主要应用于生物大分子相互作用的研究,本文采用溶液竞争法,测定了小分子吗啡与其抗体作用的结合常数K₁,并计算了吗啡抗体与吗啡化牛血清白蛋白的结合常数K₂.证明了多抗对抗原的亲和力较单抗大,并表明大分子蛋白质的存在对抗体与待测物的结合有阻碍作用.     

彩色表面等离子体共振成像初探

自主设计并建立了彩色表面等离子共振成像(Color SPRI, CSPRI)实验系统, 并结合利用自己编制的软件开展了相关研究, 成功地观测到溶液和蛋白点阵的彩色图像. 这些结果显示, CSPRI有可能成为生物分子微点阵(或生物芯片)的一种新型的彩色显示手段.