表面等离子体共振光谱技术与电化学方法联用及其应用

表面等离子体共振（Surface Plasmon Resonance,SPR）技术是利用金属薄膜光学耦合产生的物理光学现象建立的一种非常灵敏的光学分析手段. 近年发展的电化学表面等离子体共振（Electrochemical Surface Plasmon Resonance,EC-SPR）是将时间分辨表面等离子体共振光谱技术与电化学方法联用的一种新技术. 本文介绍了SPR和EC-SPR的基本原理,并重点阐述了时间分辨SPR光谱技术与电化学方法联用及应用,该技术已广泛地应用于反应动态过程研究、生物化学传感器、电极/溶液界面的表征、动力学常数的测定以及生物分子相互作用等领域.     

Present and future of surface plasmon resonance biosensors

Surface plasmon resonance (SPR) biosensors are optical sensors exploiting special electromagnetic waves—surface plasmon-polaritons—to probe interactions between an analyte in solution and a biomolecular recognition element immobilized on the SPR sensor surface. Major application areas include detection of biological analytes and analysis of biomolecular interactions where SPR biosensors provide benefits of label-free real-time analytical technology. This paper reviews fundamentals of SPR affinity biosensors and discusses recent advances in development and applications of SPR biosensors.     

表面等离子体共振传感技术在高分子研究中的应用

介绍了表面等离子体共振 (SPR)的基本原理和SPR传感器技术的应用现状 ,综述了SPR传感器在高分子科学研究中的应用和发展前景。     

表面等离子体共振技术在电化学反应过程研究中的应用

简述了表面等离子体共振(SPR)的基本原理,并综述了表面等离子体共振技术在电化学反应过程中的应用。SPR技术可以无需任何标记原位实时地检测分子间的相互作用,也可用于连续监测吸附/脱附和缔合/解离过程。表面等离子共振光谱(SPRS)与电化学技术结合可用来同时表征和处理电极/溶液的界面,在电化学掺杂/去掺杂过程、吸附/脱附反应的研究、痕量物质的检测、薄膜厚度、介电常数的测定等方面的应用已取得了很大的进展。     

Characterizing the interaction between aptamers and human IgE by use of surface plasmon resonance

Human immunoglobulin E (hIgE) is such an important protein, because of its involvement in allergic disease, that it is of significance to study the interactions between it and its recognizing elements. In this report an analytical strategy based on surface plasmon resonance (SPR) was developed to probe the pattern of interaction between hIgE and its recognizing molecules, including aptamers and antibodies. The affinity constants of hIgE for the antibody and the aptamer were compared first; the aptamer has more affinity than the antibody for human IgE. To study their pattern of interaction, three different binding approaches, including adding the antibody and the streptavidin-coupled aptamer to the sensing surface, were designed. The results showed that hIgE captured on the sensing surface could form a multivalent complex with the aptamer. An ELISA-like assay using the aptamer as both capture and detection probes was then developed. This work highlights an SPR method for characterizing the interaction between the protein and aptamers that is useful for study of biomolecular interaction patterns and binding properties. Figure Schematic diagram of the use of surface plasmon resonance for detection of the pattern of interaction of human IgE with its DNA aptamer and antibody     

表面等离子体共振生物传感器及其应用

表面等离子体共振(Surface Plasmon Resonance,SPR)生物传感器在检测生物分子特异性结合方面具有的高灵敏度、免标记及检测快速等优点,使其在过去的20年中在生命科学、医药科学、食品安全等领域取得了快速发展。本文对SPR生物传感器进行了简要介绍,并着重对其在生命科学、医药学、环境分析、食品安全等领域的应用进行了综述,最后对该领域的研究前景进行了展望。     

Surface plasmon resonance

During last decade there has been significant progress in the development of analytical techniques for evaluation of receptor-ligand iteraction. Surface plasmon resonance (SPR)-based optical biosensors are now being used extensively to defined the kinetics of wide variety of macromolecular interactions and high- and low-affinity small molecule interactions. The experimental design data analysis methods are evolving along with widespread applications in ligand fishing, microbiology, virology, host-pathogen interaction, epitope mapping and protein-, cell-, membrane-, nucleic acid-protein interactions. SPR based biosensors have strong impact on basic and applied research significantly. This brief review describes the SPR technology and few of its applications in relation to receptor-ligand interaction that has brought significant change in the methodology, analysis, interpretation, and application of the SPR technology.     

Application of surface plasmon resonance for the detection of carbohydrates,glycoconjugates, and measurement of the carbohydrate-specific interactions: A comparison with conventional analytical techniques. A critical review

Carbohydrates (glycans) and their conjugates with proteins and lipids contribute significantly to many biological processes. That makes these compounds important targets to be detected, monitored and identified. The identification of the carbohydrate content in their conjugates with proteins and lipids (glycoforms) is often a challenging task. Most of the conventional instrumental analytical techniques are time-consuming and require tedious sample pretreatment and utilising various labeling agents. Surface plasmon resonance (SPR) has been intensively developed during last two decades and has received the increasing attention for different applications, from the real-time monitoring of affinity bindings to biosensors. SPR does not require any labels and is capable of direct measurement of biospecific interaction occurring on the sensing surface. This review provides a critical comparison of modern analytical instrumental techniques with SPR in terms of their analytical capabilities to detect carbohydrates, their conjugates with proteins and lipids and to study the carbohydrate-specific bindings. A few selected examples of the SPR approaches developed during 2004–2011 for the biosensing of glycoforms and for glycan–protein affinity studies are comprehensively discussed.     

表面等离子共振-质谱法对相互作用的生物分子在10-15mol水平的微量鉴定

利用生物传感芯片质谱法(BIA/MS)对微球蛋白及其抗体的相互作用进行分析和鉴定.将微球蛋白抗体偶联到芯片上,让微球蛋白溶液流过芯片表面,然后使用“三明治”结构的微再生方法把结合的微球蛋白从芯片上洗脱下来,再对其进行酶解及质谱鉴定,在10-15mol水平得到了明确的结果.     

表面等离子体共振技术研究生物膜的进展

利用表面等离子体共振技术研究生物膜是近年来兴起的一个热门课题。文章综述了表面等离子体共振技术的基本原理、主要的成膜方式、研究进展以及发展趋势。     

实时生物分子相互作用分析技术用于链霉亲和素-生物素化抗体的层层组装研究

使用生物分子相互作用分析（Biomolecular interaction analysis,BIA)技术实时监测了在链霉素和素表面层层组装亲和素－生物素化抗体多层膜的过程，结果表明，通过链霉素和素与生物素之间的强亲和作用，能够在表面形成均一的多层膜，并用实时BIA技术求得了每层蛋白质的表面浓度，对于生物素化抗体，单层吸附表面浓度为1.32ng/mm^2；对于链霉亲和素，单层吸附表面浓度为2.93ng/mm^2。同时对蛋白质在表面的排列状态进行了探讨。     

Development of an optical biosensor assay for detection of β-lactam antibiotics in milk using the penicillin-binding protein 2x*

An assay was developed for the detection of residues of penicillins and cephalosporins in milk using a surface plasmon resonance (SPR) biosensor. The assay was based on the inhibition of the binding of digoxigenin-labelled ampicillin (DIG-AMPI) to a soluble penicillin-binding protein 2x derivative (PBP 2x*) of Streptococcus pneumoniae. Samples were incubated with PBP 2x* in a first step, whereby β-lactams in positive samples would bind to the PBP 2x*. Non-complexed PBP 2x* was then allowed to form a complex with DIG-AMPI in a second incubation step. The formed DIG-AMPI/PBP 2x*-complexes were detected in a SPR-based biospecific interaction assay (BIA) for digoxigenin with an antibody against digoxigenin immobilised on the sensor chip. Although binding of matrix components to the sensor chip (non-specific binding) occurred, benzylpenicillin, ampicillin, amoxicillin, cloxacillin, cephalexin and cefoperazone could be detected in defatted bulk raw milk samples at concentrations corresponding to the maximum residue limits (MRL) set by the European Union. The influence of matrix components on the performance of the assay was examined in more detail by analysing individual raw milk samples from 19 cows. Compared to bulk raw milk samples, individual samples showed a higher level and variation of matrix interferences. Non-specific binding could be reduced to a lower and more constant level by a heat-treatment step, a centrifugation step and the addition of carboxymethylated dextran to the samples. With this sample preparation, benzylpenicillin could be detected at MRL (4 μg kg⁻¹) in individual raw milk samples. Thus, the assay could be the basis for a screening test for routine use.     

Discourse on the utilization of polyaniline coatings for surface plasmon resonance sensing of ammonia vapor

Surface plasmon resonance spectroscopy is sensitive to near-surface (<300 nm) chemical and physical events that result in refractive index changes. The non-specific nature of the stimulus implies that chemical selectivity in SPR sensing configurations entirely relies upon the chemical recognition scheme employed. Biosensing applications commonly use surface layers composed of antibodies or enzymes for biomolecular recognition. Monitoring of volatile compounds with SPR spectroscopy, however, has not been widely discussed due to the difficulty in selectively responding to small molecules (<100 Da) in addition to the limited refractive index changes resulting from the interaction between the plasmon wave and volatile compounds.Different strategies explored thus far for sensing of small molecules have relied on optical and electrical changes of the recognition layer upon exposure to the analyte, yielding an indirect measurement. Examples of coatings used for gas-phase sensing with SPR include conducting metal oxides, polymers and organometallic dyes. Electrically conducting polymers, like polyaniline and polypyrrole, display dramatic conductivity changes in the presence of certain compounds. This property has resulted in their routine incorporation into different sensing schemes. However, application of electrically conducting polymers to SPR gas-phase sensing has been limited to a few examples, despite encouraging results.The emeraldine salt form of polyaniline (PAni) demonstrates a decreased electrical conductivity correlated to NH₃ concentration. In this contribution, PAni doped with camphorsulfonic acid (PAni-CSA) was applied to gas-phase sensing of NH₃ by way of SPR spectroscopy. Spectroscopic ellipsometry was used to determine the optical constants (n and k) for emeraldine salt and emeraldine base forms of PAni, confirming the wavelength-dependent response observed via SPR. The analytical performance of the coatings show that a limit of detection of 32 ppm NH₃ based on precision of the mass-flow controllers used and an estimated method limit of detection of ∼0.2 ppm based on three standard deviations of the blank. This is directly comparable to other, more established sensing architectures.     

Microchannel chips for the multiplexed analysis of human immunoglobulin G–antibody interactions by surface plasmon resonance imaging

We report the multiplexed, simultaneous analysis of antigen–antibody interactions that involve human immunoglobulin G (IgG) on a gold substrate by the surface plasmon resonance imaging method. A multichannel, microfluidic chip was fabricated from poly(dimethylsiloxane) (PDMS) to selectively functionalize the surface and deliver the analyte solutions. The sensing interface was constructed using avidin as a linker layer between the surface-bound biotinylated bovine serum albumin and biotinylated anti-human IgG antibodies. Four mouse anti-human IgG antibodies were selected for evaluation and the screening was achieved by simultaneously monitoring protein–protein interactions under identical conditions. Antibody–antigen binding affinities towards human immunoglobulin were quantitatively compared by employing Langmuir adsorption isotherms for the analysis of SPRi responses obtained under equilibrium conditions. We were able to identify two IgG samples with higher affinities towards the target, and the determined binding kinetics falls within the typical range of values reported in the literature. Direct measurement of proteins in serum samples by SPR imaging was achieved by developing methods to minimize nonspecific adsorption onto the avidin-functionalized surface, and a limit of detection (LOD) of 6.7 nM IgG was obtained for the treated serum samples. The combination of SPR imaging and multichannel PDMS chips offers convenience and flexibility for sensitive and label-free measurement of protein–protein interactions in complex conditions and enables high-throughput screening of pharmaceutically significant molecules. Figure Microchannel SPR imaging for protein–protein interactions     

Fe3O4 nanoparticles-enhanced SPR sensing for ultrasensitive sandwich bio-assay

Magnetic nanoparticles (MNPs) have been receiving increasing attention because of its great potentials in bioseparation. However, the separation products are difficult to be detected by general method due to their extremely small size. Here, we demonstrate that MNPs can greatly enhance the signal of surface plasmon resonance spectroscopy (SPR). Features of MNPs-aptamer conjugates as a powerful amplification reagent for ultrasensitive immunoassay are reported in this work for the first time. In order to evaluate the sensing ability of MNPs-aptamer conjugates as an amplification reagent, a sandwich SPR sensor is constructed by using thrombin as model analyte. Thrombin, captured by immobilized anti-thrombin aptamer on SPR gold film, is sensitively detected by SPR spectroscopy with a lowest detection limit of 0.017 nM after MNPs-aptamer conjugates is used as amplification reagent. At the same time, the excellent selectivity of the present biosensor is also confirmed by using three kinds of proteins (BSA, human IgM and human IgE) as controls. These results confirm that MNPs is a powerful sandwich element and an excellent amplification reagent for SPR based sandwich immunoassay and SPR has a great potential for the detection of MNPs-based bioseparation products.     

Label-free optical diagnosis of hepatitis B virus with genetically engineered fusion proteins

A simple biosensing strategy for the diagnosis of patients with hepatitis B virus (HBV) was developed. This study can be divided into two themes, both of which utilized gold-binding polypeptide (GBP) fusion proteins: HBV surface antigen PreS2 (HBsAg) detection with GBP-fused single chain antibody (GBP-ScFv) and anti-HBsAg detection with GBP-HBsAg. These GBP-fusion proteins can directly bind onto the gold surface via the high binding affinity between the GBP and the gold surface, while at the same time, orient the recognition sites toward the sample for target binding. This one-step immobilization strategy, which greatly simplifies a fabrication process as well as maintaining biological activity of the recognition elements, can be applied to optical analytical methods, such as surface plasmon resonance (SPR) and localized surface plasmon resonance (LSPR).     

A Surface Plasmon Resonance Sensor Platform Coupled with Gold Nanoparticle Probes for Unpurified Nucleic Acids Detection

In this work, a surface plasmon resonance sensor system was designed and implemented for determination of nucleic acids in unpurified samples. First, through blocking non-specific interaction sites on the sensor surface to reduce non-specific adsorption from unpurified sample matrix, it was determined that at the optimal BSA concentration of 100 µg/ml the non-specific interaction can be reduced by 50%, although improvement for direct detection of nucleic acids in unpurified sample is required. Second, bearing nonspecific adsorption onto gold films, nucleic acids adsorbed on sensor surface in unpurified sample matrix were detected through a secondary hybridization approach. Using DNA-lined AuNPs shows the new SPR sensor can be applied for the determination of target ssDNA with a detection range of 0.1–10 µM for targets in purified and 1–10 µM in unpurified samples, respectively. Results imply that the new SPR sensor system is promising for specific and convenient analysis of nucleic acids directly in unpurified samples. Development of the new SPR sensor technique can have applications in fast field diagnostics and monitoring.     

A novel method to prepare SPR sensor chips based on photografting molecularly imprinted polymer

A novel method to prepare surface plasmon resonance(SPR) sensor chips based on grafted imprinted polymer is explored. Benzophenone photografting system is used to grow molecularly imprinted polymer(MIP) films from the modified surface of gold substrate.The surface morphology and thickness of MIP films were investigated by scanning electronic microscope(SEM).The adsorption properties of sensor chip were studied by SPR spectroscopy.The results demonstrate that nano-MIP films can be constructed on the surface of gold substrate with the good adsorption of template molecules.     

表面等离子体共振技术在蛋白质-蛋白质相互作用研究中的应用

表面等离子共振(SPR)近年来迅速发展为用于分析生物分子相互作用的一项技术.该技术无需标记、特异性强、灵敏度高、样品用量小,可实现在线连续实时检测.目前SPR已被广泛应用于免疫学、蛋白质组学、药物筛选、细胞信号转导、受体/配体垂钓等领域.该文阐述了基于表面等离子体共振技术生物传感器的基本原理和技术流程,综述了SPR在蛋白质-蛋白质相互作用动力学研究、蛋白质结构及功能研究、蛋白质突变和碎片分析、信号转导中的应用以及SPR在蛋白质-蛋白质相互作用研究中的多项关键技术.指出SPR通过与光谱、电化学等多技术联用后,可以获得更加详实的信息.