SPR生物传感器的特点及其在生物特异性相互作用分析中的应用

利用表面等离子模共振技术（SPR）进行生物特异性相互作用分析（BIA）已成为现代基因工程技术中的一种先进的手段。与传统的研究方法如酶联免疫吸附测定（ELISA）相比,它具有方便快捷、灵敏度高,应用范围广,实时监控等多项特点,利用这种新型研究手段对于生命科学的基础研究。医学诊断以及治疗等方面有着十分重的意义。本粗略概括了近几年来利用SPR生物传感器进行基础研究的基本情况以及对其的改进,并简要分析了此项技术的优点以及发展前景。     

基于SPR生物传感器的免疫学检测

利用一种全新的生物大分子相互作用检测仪表面等离子激元共振（SPR）生物传感器,对乙肝表面抗原,抗体,破伤风类毒素,抗体等生物制品进行生物特异性相互作用分析（BIA）,并对其在免疫学检测上的特征进行了探讨。     

五种SPR传感芯片的再生制备及其应用

基于表面等离子体共振技术(surface plasmon resonance, SPR)的生物传感器,能够实时监测生物分子间的相互作用,且无需标记,已被广泛应用于蛋白质组学、药物研发、临床诊断、食品安全和环境监测等领域,并且显示出广阔的应用前景。传感芯片是Biacore系列仪器的核心部件,目前芯片只能从Biacore公司购买,价格昂贵,导致很多仪器利用率低下,资源处于闲置状态。阐述了用于Biacore系列仪器的五种传感芯片（J1,C1,CM5,SA和NTA芯片）的再生制备方法,并列举了应用实例,制备方法操作简单,成本低廉。通过多年的改进与优化,制备的芯片能够达到Biacore芯片同等品质。此方法的推广,将有助于推动表面等离子共振技术在各个领域的广泛应用。     

SPR生物传感器的谱之数字解析

为了实时地从ＳＰＲ谱的变化得到生物大分子相互作用的信息,作者采用等效递推法编程模拟计算,探讨了影响ＳＰＲ谱的各种因素。结果表明：在实际应用中组装在金膜表面的多层生物样品的厚度、面密度与ＳＰＲ谱峰位移ΔθＳＰＲ成正比,单个样品分子所占的面积与ΔθＳＰＲ成反比。光源波长和棱镜玻璃的色散关系对上述关系的比例常数有较大的影响;虽然作为金膜载体的盖玻片会导致ＳＰＲ谱上叠加一定频率的干涉噪音,但是可以不考虑盖玻片的厚度以及金膜厚度的影响。为了实时地从ＳＰＲ谱的变化得到生物大分子相互作用的信息,作者采用等效递推法编程模拟计算,探讨了影响ＳＰＲ谱的各种因素。结果表明：在实际应用中组装在金膜表面的多层生物样品的厚度、面密度与ＳＰＲ谱峰位移ΔθＳＰＲ成正比,单个样品分子所占的面积与ΔθＳＰＲ成反比。光源波长和棱镜玻璃的色散关系对上述关系的比例常数有较大的影响;虽然作为金膜载体的盖玻片会导致ＳＰＲ谱上叠加一定频率的干涉噪音,但是可以不考虑盖玻片的厚度以及金膜厚度的影响。     

表面等离子体激元共振与生物分子相互作用分析

对表面等离子体激元共振（surface plasmon resonance, SPR）的原理和在生物学研究方面的应用进行了综述.这种技术可以直接原位、实时地跟踪生物学实验研究系统,而不需要附加参数如进行标记等手段,具有高敏感性,也可以连续监测吸附或解吸附过程,目前有关的应用涉及到生物学结合分析、动力学及亲和力测定、免疫识别研究、结构与活性研究和核酸研究等多个领域.     

表面等离子体共振技术在分子生物学中的应用

表面等离子体共振(SPR)技术可以实时、原位地测定生物分子间的相互作用而无需任何标记,可以连续监测吸附和解离过程,并可以进行多组分复合物的相互作用的研究。SPR技术在DNA的复制和转录、DNA的修复、核酸与药物的作用以及肽库和抗体库的筛选等分子生物学领域的应用研究取得了令人瞩目的进展,显示了常规技术无法比拟的优越性。     

压电生物传感器与光生物传感器

压电(Piezoelectronic,PZ)生物传感器和光生物传感器(Optical Biosensors)是两类新型的生物传感器,本文简要阐述它们的原理、特点和研究进展。关键词:压电生物传感器,生物光极,SPR。     

偏振干涉角度调制SPR传感技术研究及应用进展

针对一种新兴生物检测方法——表面等离子体波共振(SPR)技术,文中SPR传感系统采用偏振干涉和角度调制方案,使SPR传感灵敏度与光复反射系数的模和相位都相关,从而实现较大线形范围内的高灵敏测量。同时开展了该SPR传感系统在环保领域的应用研究,SPR共振信号可实时随甲烷含量线性改变,气体检测灵敏度达到1 070ppm,实验结果验证了这种SPR传感技术的检测性能并显示了其在环保监测领域的应用潜力。     

基于表面等离子体共振和电化学联用的DNA传感器研究进展

表面等离子共振(surface plasmon resonance,SPR)技术旨在检测物体表面附近折射率的变化,其特点是无标记、实时、灵敏和快速,该技术多用于研究分子的相互作用,包括动力学、效率常数和大分子构象变化等。电化学(electrochemical,EC)技术是一项用于定性定量研究电子转移、物质氧化还原、界面吸附等过程的成熟技术,具有简单、低成本和设备小型化的优点。现有的DNA杂交技术,例如光学、电化学或压电转导技术,主要关注于提高DNA杂交检测系统的选择性和灵敏度。传统的SPR在DNA分析方面,由于无法测量折射率的极小变化而在超灵敏检测中的应用受到限制。因此,随着纳米材料的研发和联用技术的飞速发展,SPR与EC联用的生物传感器研究越来越成为人们关注的热点。近年来,关于SPR和EC联用在DNA检测方面的综述鲜有报道。对SPR和EC检测DNA的技术原理、联用方法、应用进展等方面作出了简要的介绍,以期为表面等离子共振和电化学联用的DNA传感器相关研究提供参考。     

SPR studies of carbohydrate-lectin interactions as useful tool for screening on lectin sources

Lectins are proteins or glycoproteins from plants, animals or microorganisms, which typically bind specifically to sugar residues, e.g., located in cell walls or membranes. This reaction might change the physiology of the cell wall and influences the metabolism inside the cell. Some lectins of plants stimulate the immune system by unspecific activation of T-cells or influence cell division; others cause agglutination of cells (e.g., erythrocytes) and are therefore from therapeutic interest.

In a new approach, biomolecular interaction analysis (BIA) was utilised for a screening program on lectins. The BIA has been done by surface plasmon resonance (SPR). The system can be used either for characterisation of lectin-binding domains or for a screening on lectins from natural sources. Several lectin-binding surfaces on the basis of SPR have been established.     

Biosensor detection of triplex formation by modified oligonucleotides

Due to the instability of DNA oligonucleotides in biological solutions, antisense or antigene therapies aimed at modulation of specific gene expression will most likely require the use of oligonucleotides with modified backbones. Here, we examine the use of a surface plasmon resonance biosensor (BIAcore) to compare triplex-directed binding of modified oligonucleotides targeted to a region of the murine c-myc promoter. We describe optimization of experimental conditions to minimize nonspecific interactions between the oligonucleotides and the sensor chip surface, and the limitations imposed by certain backbones and sequence types. The abilities of pyrimidine oligonucleotides with various modified backbones to form specific triple helices with an immobilized hairpin duplex were readily determined using the biosensor. Modification of the third-strand oligonucleotide with RNA or 2(')-O-methyl RNA was found to enhance triplex formation, whereas phosphorothioate or phosphotriester substitutions abrogated it. A comparison of these results to DNase I footprinting experiments using the same oligonucleotides showed complete agreement between the two sets of data.     

基于表面等离子共振的新型生物传感技术及其在生命科学中的应用

生物分子的活性功能是通过分子之间的相互作用来体现的,了解这种相互作用的过程对于生命科学领域的研究及揭示生命发生发展的基本机制具有重要的意义。基于表面等离子共振(surface plasmon resonance,SPR)的新型生物传感技术——BIAcore(biomolecular interaction analysis)是研究生物分子相互作用的理想工具。它可以实时跟踪检测生物分子间结合、解离的整个过程,已被广泛应用于蛋白质组学、信号转导、新药开发、遗传学分析和食品检测等领域,并且显示出广阔的应用前景。     

Molecular interactions in the insulin-like growth factor (IGF) axis: a surface plasmon resonance (SPR) based biosensor study

This review describes a comprehensive analysis of a surface plasmon resonance (SPR)-based biosensor study of molecular interactions in the insulin-like growth factor (IGF) molecular axis. In this study, we focus on the interaction between the polypeptide growth factors IGF-I and IGF-II with six soluble IGF binding proteins (IGFBP 1-6), which occur naturally in various biological fluids. We have describe the conditions required for the accurate determination of kinetic rate constants for these interactions and highlight the experimental and theoretical pitfalls, which may be encountered in the early stages of such a study. We focus on IGFBP-5 and describe a site-directed mutagenesis study, which examines the contribution of various residues in the protein to high affinity interaction with IGF-I and -II. We analyse the interaction of IGFBP-5 (and IGFBP-3) with heparin and other biomolecules and describe experiments, which were designed to monitor multi-protein complex formation in this molecular axis.     

A global benchmark study using affinity-based biosensors

To explore the variability in biosensor studies, 150 participants from 20 countries were given the same protein samples and asked to determine kinetic rate constants for the interaction. We chose a protein system that was amenable to analysis using different biosensor platforms as well as by users of different expertise levels. The two proteins (a 50-kDa Fab and a 60-kDa glutathione S-transferase [GST] antigen) form a relatively high-affinity complex, so participants needed to optimize several experimental parameters, including ligand immobilization and regeneration conditions as well as analyte concentrations and injection/dissociation times. Although most participants collected binding responses that could be fit to yield kinetic parameters, the quality of a few data sets could have been improved by optimizing the assay design. Once these outliers were removed, the average reported affinity across the remaining panel of participants was 620 pM with a standard deviation of 980 pM. These results demonstrate that when this biosensor assay was designed and executed appropriately, the reported rate constants were consistent, and independent of which protein was immobilized and which biosensor was used.     

High-resolution characterization of antibody fragment/antigen interactions using Biacore T100

A Biacore T100 optical biosensor was used to characterize the binding kinetics of a panel of antigen binding fragments (Fabs) directed against the PcrV protein from Pseudomonas aeruginosa. PcrV protein forms part of the type III secretion system complex of this opportunistic pathogen. We demonstrate that the biosensor response data for each Fab collected from three different surface densities of the antigen could be fit globally to a simple 1:1 interaction model. Importantly, we found that the Fabs with the slowest dissociation rate provided the best protection in cell cytotoxicity studies. To further characterize the Fab interactions, binding data were automatically acquired at different temperatures and under different buffer conditions. The comprehensive characterization of these Fabs shows how Biacore T100 can be used to complement protein therapeutic discovery programs from basic research to the selection of therapeutic candidates.     

Sensitivity enhancement of spectral surface plasmon resonance biosensors for the analysis of protein arrays

A novel method for sensitivity enhancement of spectral surface plasmon resonance (SPR) biosensors was presented by reducing the refractive index of the sensing prism in the analysis of protein arrays. Sensitivity of spectral SPR biosensors with two different prisms (BK-7, fused silica) was analyzed by net shifts of resonance wavelength for specific interactions of GST–GTPase binding domain of p21-activated kinase-1 and anti-GST on a mixed thiol surface. Sensitivity was modulated by the refractive index of the sensing prism of the spectral SPR biosensors with the same incidence angle. The sensitivity of a spectral SPR biosensor with a fused silica prism was 1.6 times higher than that with a BK-7 prism at the same incidence angle of 46.2°. This result was interpreted by increment of the penetration depth correlated with evanescent field intensity at the metal/dielectric interface. Therefore, it is suggested that sensitivity enhancement is readily achieved by reducing the refractive index of the sensing prism of spectral SPR biosensors to be operated at long wavelength ranges for the analysis of protein arrays.     

A fusion protein expression analysis using surface plasmon resonance imaging

A surface plasmon resonance (SPR) imaging system was constructed and used to detect the affinity-tagged recombinant proteins expressed in Escherichia coli. With regards to model proteins, the hexahistidine-ubiquitin-tagged human growth hormone (His(6)-Ub-hGH), glutathione S-transferase-tagged human interleukin-6 (GST-hIL6), and maltose-binding protein-tagged human interleukin-6 (MBP-hIL6) expressed in E. coli were analyzed. The cell lysates were spotted on gold thin films coated with 11-mercaptoundecanol (MUOH)/dextran derivatized with Ni(II)-iminodiacetic acid (IDA-Ni(II)), glutathione, or cyclodextrin. After a brief washing of the gold chip, SPR imaging measurements were carried out in order to detect the bound affinity-tagged fusion proteins. Using this new approach, rapid high-throughput expression analysis of the affinity-tagged proteins were obtained. The SPR imaging protein chip system used to measure the expression of affinity-tagged proteins in a high-throughput manner is expected to be an attractive alternative to traditional laborious and time-consuming methods, such as SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blots.     

Ca-asp bound X-ray structure and inhibition of Bacillus anthracis dihydroorotase (DHOase)

Dihydroorotase (DHOase) is the third enzyme in the de novo pyrimidine synthesis pathway and is responsible for the reversible cyclization of carbamyl-aspartate (Ca-asp) to dihydroorotate (DHO). DHOase is further divided into two classes based on several structural characteristics, one of which is the length of the flexible catalytic loop that interacts with the substrate, Ca-asp, regulating the enzyme activity. Here, we present the crystal structure of Class I Bacillus anthracis DHOase with Ca-asp in the active site, which shows the peptide backbone of glycine in the shorter loop forming the necessary hydrogen bonds with the substrate, in place of the two threonines found in Class II DHOases. Despite the differences in the catalytic loop, the structure confirms that the key interactions between the substrate and active site residues are similar between Class I and Class II DHOase enzymes, which we further validated by mutagenesis studies. B. anthracis DHOase is also a potential antibacterial drug target. In order to identify prospective inhibitors, we performed high-throughput screening against several libraries using a colorimetric enzymatic assay and an orthogonal fluorescence thermal binding assay. Surface plasmon resonance was used for determining binding affinity (K_D) and competition analysis with Ca-asp. Our results highlight that the primary difference between Class I and Class II DHOase is the catalytic loop. We also identify several compounds that can potentially be further optimized as potential B. anthracis inhibitors.