SPR技术与功能基因组和蛋白质组研究

表面等离子体共振(surface plasmon resonance,SPR)依据光学—介质相互作用原理建立,属于实时和非标记的测试方法。SPR方法在研究分子间相互作用方面具有其独特的优势,其非标记和实时检测以及可以进行动力学分析的特点,给研究生物大分子的相互作用提供了诱人的解决方案。近来,随着SPR成像技术和SPR芯片制备技术的进展,将为功能基因组学和蛋白质组学研究提供重要的新的技术平台。     

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技术分析生物分子相互作用的研究方法

生物分子的活性功能是通过分子之间的相互作用来实现的,了解这种相互作用的关系时生命科学的研究及揭示生命发生发展的基本机制具有着重要的意义.基于表面等离子共振(SPR)的分析分子相互作用(BIA)的技术是新型的生物传感技术,其无需标、能实时跟踪检测生物分子间结合、解离的整个过程,通过分析传感图谱获取分子相互作用的模式和动力学常数等方面的信息.SPR是研究生物分子相互作用的强有力工具,SPR技术已被广泛应用于生命科学领域的研究,并且显示出广阔的应用前景.概述了SPR技术原理、分析方法及其评述了其存在的问题.     

SPR技术在免疫学研究中的应用

表面等离子共振(Surface Plasmon Resonance,SPR)技术是研究生物分子相互作用的强有力工具之一,该技术使生物分子之间相互作用的实时检测成为可能,并且灵敏度高、无需标记.通过分析传感图谱及分子相互作用的响应值获取分子相互作用的模式和动力学常数等方面的信息,并且获得的信息是能够定性和定量.SPR技术现在已广泛应用于生物、化学、免疫学研究及新药开发等领域.本文主要就SPR技术在免疫学研究中抗体活性检测、抗原表位预测等方面的应用进行了综述.     

SSB蛋白与ssDNA相互作用的动力学和可视化研究

研究大肠杆菌单链结合蛋白(single-stranded DNA-binding protein,SSB)与单链DNA(single-stranded DNA,ssDNA)的相互作用对于了解其在DNA复制、重组和修复中的作用是非常重要的。通过表面等离子共振技术(surface plasmon resonance,SPR)得到了在有、无镁离子的情况下,SSB与ssDNA两者的平衡解离常数(equilibrium dissociation constant,KD)分别为9.67×10-7M和4.79×10-7M,阐明了镁离子对于两者作用形式的影响。利用原子力显微镜技术分别观察SSB蛋白、ssDNA和SSB-ssDNA复合物的成像,为下一步研究SSB在DNA代谢中作用模式的单分子可视化奠定了基础。     

表面等离子体共振技术在药物研究中的应用

表面等离子体共振(surface plasmon resonance,SPR)技术作为一种新型的免标记、实时在线研究生物分子间相互作用的高灵敏传感技术,已经在生命科学领域中得到了大量应用。该文简要介绍了SPR生物传感器的基本原理,重点评述了其在新药筛选和药物作用机制方面的研究进展,并对其前景进行了展望。     

蓖麻毒素与其单克隆抗体相互作用动力学研究

表面等离子体激元共振(SPR)是一种可微量、实时、动态地监测生物分子相互作用的生物传感技术。蓖麻毒素为核糖体失活蛋白,具有很强的细胞毒性作用。通过SPR技术研究了两种抗蓖麻毒素的单克隆抗体C5、D12与蓖麻毒素相互作用的动力学,计算出两者的亲和常数分别为2.49×108mol-1·L和7.9×108mol-1·L,并对两种抗体的抗原表位进行了分析。     

利用表面等离子共振传感器检测苏云金芽孢杆菌cry1F蛋白

目的：建立检测苏云金芽孢杆菌（Bt）crylF蛋白的表面等离子共振（SPR）传感器方法。方法：采用SPR检测技术,利用生物分子相互作用分析原理,在金表面修饰特异性单克隆抗体,对crylF蛋白的检测进行研究。结果：该方法可以较好地检测到crylF蛋白,最低检测限可达10ng／mL,并且具有很好的特异性。结论：SPR检测方法的重复性较好,灵敏度高,目前可用于crylF蛋白的定性检测,为crylF蛋白及其他Bt蛋白的检测提供了新方法,在检测转Bt基因植物方面具有广阔的应用前景。     

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。     

SARS-CoV核蛋白和宿主细胞相互作用的初步研究

寻找与SARS-CoV核蛋白相互作用的宿主细胞蛋白,从而探索SARS-CoV的致病机理。可溶性表达SARS-CoV核蛋白,利用His标签和离子交换层析对表达的蛋白进行了纯化,获得较纯的可溶性核蛋白。再将SPR/BIA技术与MALDI-TOF MS技术结合起来,使用SPR生物传感芯片作为亲和吸附的表面,分别捕获2BS细胞和A549细胞裂解液中与SARS-CoV核蛋白相互作用的细胞蛋白,收集足够量的相互作用蛋白,再利用MALDI-TOF-MS分析获得蛋白的性质。结果鉴定出与SARS-CoV核蛋白相互作用的蛋白:26S蛋白酶调节亚单位S10B(蛋白酶体亚单位p42)(蛋白酶体26S亚单位ATPase 6)(P62333),属于泛素/蛋白酶体系统;目前国内外尚未见类似报道。此研究初步发现了一种与SARS-CoV核蛋白在细胞外相互作用的蛋白,但这种相互作用在SARS-CoV感染及SARS的发生发展中发挥的作用还有待于深入研究和探索。     

Label-enhanced surface plasmon resonance applied to label-free interaction analysis of small molecules and fragments

Surface plasmon resonance (SPR) is a well-established method for studying interactions between small molecules and biomolecules. In particular, SPR is being increasingly applied within fragment-based drug discovery; however, within this application area, the limited sensitivity of SPR may constitute a problem. This problem can be circumvented by the use of label-enhanced SPR that shows a 100-fold higher sensitivity as compared with conventional SPR. Truly label-free interaction data for small molecules can be obtained by applying label-enhanced SPR in a surface competition assay format. The enhanced sensitivity is accompanied by an increased specificity and inertness toward disturbances (e.g., bulk refractive index disturbances). Label-enhanced SPR can be used for fragment screening in a competitive assay format; the competitive format has the added advantage of confirming the specificity of the molecular interaction. In addition, label-enhanced SPR extends the accessible kinetic regime of SPR to the analysis of very fast fragment binding kinetics. In this article, we demonstrate the working principles and benchmark the performance of label-enhanced SPR in a model system—the interaction between carbonic anhydrase II and a number of small-molecule sulfonamide-based inhibitors.     

Label-free analysis of biomolecular interactions using SPR imaging

Surface plasmon resonance (SPR) is a label-free detection method by which molecular interactions may be analyzed on a surface. Binding data are collected in real time, allowing the determination of interaction kinetics. SPR imaging (SPRi), the focus of this review, improves upon the efficiency of SPR by facilitating analysis of multiple interactions simultaneously. Here we summarize the principles of SPRi, provide examples of how SPRi arrays can be fabricated, and illustrate the utility of SPRi through example applications from the fields of proteomics, genomics and bioengineering.     

Fab fragments imprinted SPR biosensor for real-time human immunoglobulin G detection

F(ab) fragments imprinted surface plasmon resonance (SPR) chip was prepared for the real-time detection of human immunoglobulin G (IgG). In order to attach polymerization precursor on SPR chip, the SPR chip surface was modified with allyl mercaptan. F(ab) fragments of the IgG molecules were prepared by papain digestion procedure and collected by fast protein liquid chromatography (FPLC) system using Hi-Trap_r Protein A FF column. The collected F(ab) fragments were complexed with histidine containing specific monomer, N-methacryloyl-l-histidine methyl ester (MAH). Molecular imprinted polymeric nanofilm was prepared on SPR chip in the presence of ethylene glycol dimethacrylate and 2-hydroxyethylmethacrylate. The template molecules, F(ab) fragments, were removed from the polymeric nanofilm using 1M NaCl solution (pH: 7.4, phosphate buffer system). The molecular imprinted SPR chip was characterized by contact angle, atomic force microscopy and Fourier transform infrared spectroscopy. By the real-time IgG detection studies carried out using aqueous IgG solutions in different concentrations, the kinetics and isotherm parameters of the molecular imprinted SPR chip-IgG system were calculated. To show selectivity and specificity of the molecular imprinted SPR chip, competitive kinetic analyses were performed using bovine serum albumin (BSA), IgG, F(ab) and F(c) fragments in singular and competitive manner. As last step, IgG detection studies from human plasma were performed and the measured IgG concentrations were well matched with the results determined by enzyme-linked immunosorbent assay (ELISA). The results obtained with the molecular imprinted SPR chip were well fitted to Langmuir isotherm and the detection limit was found as 56 ng/mL. In the light of the results, we can conclude that the proposed molecular imprinted SPR chip can detect IgG molecules from both aqueous solutions and complex natural samples.     

SPR生物传感器及其应用进展

基于表面等离子体共振 (SPR)技术的光学生物传感器是进行生物分子相互作用分析的一种先进手段。与传统的超速离心、荧光法等相比 ,它具有实时检测、无需标记、耗样最少等特点 ,在药物筛选、临床诊断、食物及环境监控和膜生物学等领域中的新兴应用日益扩大 ,并且已成为生命科学和制药研究的一种标准的生物物理学工具。综述了近几年国际上生物传感器的应用进展情况 ,并简要展望了该技术的发展和应用前景     

Surface plasmon resonance study of the molecular recognition between polymerase and DNA containing various mismatches and conformational changes of DNA-protein complexes

Although surface plasmon resonance (SPR) biosensor technique has been used to study protein-protein interactions and to detect conformational changes of proteins, it has not been shown whether the SPR biosensor can be used to study a complex kinetic system such as the protein-DNA binding, which sometimes involves several binding steps as well as dynamic conformational changes of the complexes. In this study, we have used SPR biosensor and T7 polymerase as the model system to study the interactions of the polymerase with a series of DNA template-primer duplexes containing different number of mismatches and GC contents at various positions near the primer 3'-end. In general, the binding constants measured by the SPR are several magnitudes smaller than those determined in solution, indicating the limitation of the surface-based technique for measuring solution-based interactions. However, the distinct polymerase binding profiles obtained for DNA duplexes differed by as low as a single mismatch suggest that the SPR data can be used for relative comparison purpose among a set of experiments carried out under identical conditions. The successful fitting of the binding profiles using the established translocation model also demonstrated that SPR can be used to monitor conformational changes, as well as to derive relative kinetic values, within a complicated DNA-protein interaction system. The results also demonstrated that SPR biosensor may be used as a sensitive technique for studying molecular recognition events, such as single-base discrimination involved in protein-DNA interactions.     

Temperature-Regulated Surface Plasmon Resonance Imaging System for Bioaffinity Sensing

We describe a temperature-regulated surface plasmon resonance (SPR) imaging biosensor in this article. The sample temperature can be regulated for specific requirements of the bioaffinity sensing, and stabilized to suppress the measurement noise caused by temperature fluctuations. The water thermo optic coefficient is measured to test the temperature regulation performance. The protein interaction is monitored to demonstrate the feasibility of this system for real-time biomolecular interaction analysis. This temperature-regulated SPR imaging biosensor can be readily implemented by adding the common water path and peristaltic pump to the conventional SPR imaging system, which may provide an economical and convenient scheme to improve the analysis accuracy and quality of bioaffinity sensing using SPR sensing platform.     

Real-time monitoring of odorant-induced cellular reactions using surface plasmon resonance

Surface plasmon resonance (SPR) is a powerful technique for measuring molecular interaction in real-time. SPR can be used to detect molecule to cell interactions as well as molecule to molecule interactions. In this study, the SPR-based biosensing technique was applied to real-time monitoring of odorant-induced cellular reactions. An olfactory receptor, OR I7, was fused with a rho-tag import sequence at the N-terminus of OR I7, and expressed on the surface of human embryonic kidney (HEK)-293 cells. These cells were then immobilized on a SPR sensor chip. The intensity of the SPR response was linearly dependent on the amount of injected odorant. Among all the aldehyde containing odorants tested, the SPR response was specifically high for octanal, which is the known cognate odorant for the OR I7. This SPR response is believed to have resulted from intracellular signaling triggered by the binding of odorant molecules to the olfactory receptors expressed on the cell surface. This SPR system combined with olfactory receptor-expressed cells provides a new olfactory biosensor system for selective and quantitative detection of volatile compounds.     

Metallic Nanowire Array–Polymer Hybrid Film for Surface Plasmon Resonance Sensitivity Enhancement and Spectral Range Enlargement

In this paper, the coupling interaction is investigated between a metallic nanowire array and a metal film under the Kretschmann condition. The plasmonic multilayer is composed of a metallic nanowire array embedded in a polymer layer positioned above a metal film, exploiting the classical surface plasmon resonance (SPR) configuration. We analyze the influence of various structural parameters of the metallic nanowire array on the SPR spectrum of thin metal film. The results show that the coupling interactions of nanowires with the metal film can greatly affect SPR resonance wavelength and increase SPR sensitivity. The coupling strength of metallic nanowire array and metal film also impacts resonance wavelength, which can be used to adjust SPR range but have little effect on its sensitivity. The results are confirmed using a dipole coupling resonance model of metallic nanowire. We demonstrated that this nanostructured hybrid structure can be used for high sensitivity SPR monitoring in a large spectral range, which is important for advanced SPR measurement including fiber-optic SPR sensing technology.     

Surface plasmon resonance for high-throughput ligand screening of membrane-bound proteins

Technologies based on surface plasmon resonance (SPR) have allowed rapid, label-free characterization of protein-protein and protein-small molecule interactions. SPR has become the gold standard in industrial and academic settings, in which the interaction between a pair of soluble binding partners is characterized in detail or a library of molecules is screened for binding against a single soluble protein. In spite of these successes, SPR is only beginning to be adapted to the needs of membrane-bound proteins which are difficult to study in situ but represent promising targets for drug and biomarker development. Existing technologies, such as BIAcoreTM, have been adapted for membrane protein analysis by building supported lipid layers or capturing lipid vesicles on existing chips. Newer technologies, still in development, will allow membrane proteins to be presented in native or near-native formats. These include SPR nanopore arrays, in which lipid bilayers containing membrane proteins stably span small pores that are addressable from both sides of the bilayer. Here, we discuss current SPR instrumentation and the potential for SPR nanopore arrays to enable quantitative, high-throughput screening of G protein coupled receptor ligands and applications in basic cellular biology.