Characterization of the surfaces generated by liposome binding to the modified dextran matrix of a surface plasmon resonance sensor chip

The dextran matrix of a surface plasmon resonance (SPR) sensor chip modified with hydrophobic residues (BIAcore sensor chip L1) provides an ideal substrate for liposome adsorption. Liposomes of different lipid compositions are captured on the sensor chips by inserting these residues into the liposome membrane, thereby generating stable lipid surfaces. To gain a more detailed understanding of these surfaces, and to prove whether the liposomes stay on the matrix as single particles or form a continuous lipid layer by liposome fusion, we have investigated these materials, using atomic force microscopy (AFM) and fluorescence microscopy. Force measurements with AFM probes functionalized with bovine serum albumin (BSA) were employed to recognize liposome adsorption. Analysis of the maximal adhesive force and adhesion energy reveals a stronger interaction between BSA and the dextran matrix compared to the lipid-covered surfaces. Images generated using BSA-coated AFM tips indicated a complete and homogeneous coverage of the surface by phospholipid. Single liposomes could not be detected even at lower lipid concentrations, indicating that the liposomes fuse and form a lipid bilayer on the dextran matrix. Experiments with fluorescently labeled liposomes concurred with the AFM studies. Surfaces incubated with liposomes loaded with TRITC-labeled dextran showed no fluorescence, indicating a complete release of the encapsulated dye. In contrast, surfaces incubated with liposomes containing a fluorescently labeled lipid showed fluorescence.     

Protein-heparin interactions measured by BIAcore 2000 are affected by the method of heparin immobilization

Surface plasmon resonance (SPR) biosensors such as the BIAcore 2000 are a useful tool for the analysis of protein-heparin interactions. Generally, biotinylated heparin is captured on a streptavidin-coated surface to create heparinized surfaces for subsequent binding analyses. In this study we investigated three commonly used techniques for the biotinylation of heparin, namely coupling through either carboxylate groups or unsubstituted amines along the heparin chain, or through the reducing terminus of the heparin chain. Biotinylated heparin derivatives were immobilized on streptavidin sensor chips and several heparin-binding proteins were examined. Of the surfaces investigated, heparin attached through the reducing terminus had the highest binding capacity, and in some cases had a higher affinity for the proteins tested. Heparin immobilized via intrachain bare amines had intermediate binding capacity and affinity, and heparin immobilized through the carboxylate groups of uronic acids had the lowest capacity for the proteins tested. These results suggest that immobilizing heparin to a surface via intrachain modifications of the heparin molecule can affect the binding of particular heparin-binding proteins.     

Surface plasmon resonance spectroscopy in the study of membrane-mediated cell signalling.

Peptide-membrane interactions contribute to many important biological processes such as cellular signaling, protein trafficking and ion-channel formation. During receptor-mediated signalling, activated intracellular signalling molecules are often recruited into receptor-induced signaling complexes at the cytoplasmic surface of the cell membrane. Such recruitment can depend upon protein-protein and protein-lipid interactions as well as protein acylation. A wide variety of biophysical techniques have been combined with the use of model membrane systems to study these interactions and have provided important information on the relationship between the structure of these proteins involved in cell signalling and their biological function. More recently, surface plasmon resonance (SPR) spectroscopy has also been applied to the study of biomembrane-based systems using both planar mono- or bilayers or liposomes. This article provides an overview of these recent applications, which demonstrate the potential of SPR to enhance our molecular understanding of membrane-mediated cellular signalling.     

Surface plasmon resonance assay for real-time monitoring of somatic coliphages in wastewaters

The surface plasmon resonance (SPR) technique is a well-established method for the measurement of molecules binding to surfaces and the quantification of binding constants between surface-immobilized proteins and proteins in solution. In this paper we describe an extension of the methodology to study bacteriophage-bacterium interactions. A two-channel microfluidic SPR sensor device was used to detect the presence of somatic coliphages, a group of bacteriophages that have been proposed as fecal pollution indicators in water, using their host, Escherichia coli WG5, as a target for their selective detection. The bacterium, E. coli WG5, was immobilized on gold sensor chips using avidin-biotin and bacteriophages extracted from wastewater added. The initial binding of the bacteriophage was observed at high concentrations, and a separate, time-delayed cell lysis event also was observed, which was sensitive to bacteriophage at low concentrations. As few as 1 PFU/ml of bacteriophage injected into the chamber could be detected after a phage incubation period of 120 min, which equates to an approximate limit of detection of around 10(2) PFU/ml. The bacteriophage-bacterium interaction appeared to cause a structural change in the surface-bound bacteria, possibly due to collapse of the cell, which was observed as an increase in mass density on the sensor chip. These results suggest that this methodology could be employed for future biosensor technologies and for quantification of the bacteriophage concentration.     

Investigation of selective protein immobilization on charged protein array by wavelength interrogation-based SPR sensor

We have investigated the use of multilayer films of polyelectrolytes as selective surfaces to analyze protein interactions with a self-assembled SPR wavelength-shift sensor. Charged arrays were prepared by alternating adsorption of the charged polyelectrolytes, poly(diallyldimethylammonium chloride) (PDDA) and poly(sodium 4-styrenesulfonate) (PSS). Multilayer formation was monitored with the SPR wavelength-shift sensor and a Spreeta SPR sensor. Protein immobilization on the charged surfaces, which was also analyzed by the SPR sensors, was dependent on the pI of the proteins. Tissue transglutaminase (tTGase) and beta-galactosidase (pIs, 5.1 and 5.3, respectively) were preferentially bound to the positively charged PDDA surface, whereas lysozyme (pI, 11.0) was selectively bound to the negatively charged PSS surface. Immobilization of tTGase on the PDDA surface was also dependent on the buffer pH. The interaction of tTGase with RhoA(V14), a constitutively active form of Rho, could be detected on the charged arrays with the wavelength-shift sensor. The arrays could be reutilized at least 5 times. Thus, it is likely that charged surfaces, assembled by the layer-by-layer method using polyelectrolytes, will prove useful for preparing selective protein arrays.     

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。     

Surface plasmon resonance protein sensor using Vroman effect

We report a new surface plasmon resonance (SPR) protein sensor using the Vroman effect for real-time, sensitive and selective detection of protein. The sensor relies on the competitive nature of protein adsorption onto the surface, directly depending upon protein's molecular weight. The sensor uses SPR for highly sensitive biomolecular interactions detection and the Vroman effect for highly selective detection. By using the Vroman effect we bypass having to rely on bio-receptors and their attachment to transducers, a process known to be complex and time-consuming. The protein sensor is microfabricated to perform real-time protein detection using four different proteins including aprotinin (0.65kDa), lysozyme (14.7kDa), streptavidine (53kDa), and isolectin (114kDa) on three different surfaces, namely a bare-gold surface and two others modified by OH- and COOH-terminated self-assembled monolayer (SAM). The real-time adsorption and displacement of the proteins are observed by SPR and evaluated using an atomic force microscope (AFM). The sensor can distinguish proteins of at least 14.05kDa in molecular weight and demonstrate a very low false positive rate. The protein detector can be integrated with microfluidic systems to provide extremely sensitive and selective analytical capability.     

Surface plasmon resonance for monitoring the interaction of Potato virus Y with monoclonal antibodies

Surface plasmon resonance (SPR)-based biosensors have been widely utilized for measuring interactions of a variety of molecules. Fewer examples include higher biological entities such as bacteria and viruses, and even fewer deal with plant viruses. Here, we describe the optimization of an SPR sensor chip for evaluation of the interaction of the economically relevant filamentous Potato virus Y (PVY) with monoclonal antibodies. Different virus isolates were efficiently and stably bound to a previously immobilized polyclonal antibody surface, which remained stable over subsequent injection regeneration steps. The ability of the biosensor to detect and quantify PVY particles was compared with ELISA and RT-qPCR. Stably captured virus surfaces were successfully used to explore kinetic parameters of the interaction of a panel of monoclonal antibodies with two PVY isolates representing the main viral serotypes N and O. In addition, the optimized biosensor proved to be suitable for evaluating whether two given monoclonal antibodies compete for the same epitope within the viral particle surface. The strategy proposed in this work can help to improve existing serologic diagnostic tools that target PVY and will allow investigation of the inherent serological variability of the virus and exploration for new interactions of PVY particles with other proteins.     

Lipid-induced conformation and lipid-binding properties of cytolytic and antimicrobial peptides: determination and biological specificity

While antimicrobial and cytolytic peptides exert their effects on cells largely by interacting with the lipid bilayers of their membranes, the influence of the cell membrane lipid composition on the specificity of these peptides towards a given organism is not yet understood. The lack of experimental model systems that mimic the complexity of natural cell membranes has hampered efforts to establish a direct correlation between the induced conformation of these peptides upon binding to cell membranes and their biological specificities. Nevertheless, studies using model membranes reconstituted from lipids and a few membrane-associated proteins, combined with spectroscopic techniques (i.e. circular dichroism, fluorescence spectroscopy, Fourier transform infra red spectroscopy, etc.), have provided information on specific structure-function relationships of peptide-membrane interactions at the molecular level. Reversed phase-high performance chromatography (RP-HPLC) and surface plasmon resonance (SPR) are emerging techniques for the study of the dynamics of the interactions between cytolytic and antimicrobial peptides and lipid surfaces. Thus, the immobilization of lipid moieties onto RP-HPLC sorbent now allows the investigation of peptide conformational transition upon interaction with membrane surfaces, while SPR allows the observation of the time course of peptide binding to membrane surfaces. Such studies have clearly demonstrated the complexity of peptide-membrane interactions in terms of the mutual changes in peptide binding, conformation, orientation, and lipid organization, and have, to a certain extent, allowed correlations to be drawn between peptide conformational properties and lytic activity.     

Surface plasmon resonance detection of blood coagulation and platelet adhesion under venous and arterial shear conditions

A surface plasmon resonance (SPR) based flow chamber device was designed for real time detection of blood coagulation and platelet adhesion in platelet rich plasma (PRP) and whole blood. The system allowed the detection of surface interactions throughout the 6mm length of the flow chamber. After deposition of thromboplastin onto a section of the sensor surface near the inlet of the flow chamber, coagulation was detected downstream of this position corresponding to a SPR signal of 7 to 8 mRIU (7 to 8 ng/mm2). A nonmodified control surface induced coagulation 3.5 times slower. Platelet adhesion to gold and fibrinogen coated surfaces in the magnitude of 1.25 and 1.66 mRIU was also shown with platelets in buffer, respectively. SPR responses obtained with PRP and whole blood on surfaces that were methylated or coated with von Willebrand factor (vWF), fibrinogen, or collagen, coincided well with platelet adhesion as observed with fluorescence microscopy in parallel experiments. The present SPR detection equipped flow chamber system is a promising tool for studies on coagulation events and blood cell adhesion under physiological flow conditions, and allows monitoring of short-range surface processes in whole blood.     

一种葡聚糖芯片的再生方法、表征及其应用*

表面等离子体共振（surface plasmon resonance, SPR）生物传感器,作为一种适时快捷,无需标记的生物分子相互作用研究工具,已广泛应用于生物化学分析与研究。羧甲基化葡聚糖修饰的CM5传感芯片是Biacore 系列仪器应用最为普遍的核心部件,目前CM5芯片主要从法玛西亚公司购买,价格昂贵,且一旦共价交联的受体分子失活,就不能重复利用。阐述了一种简便、低成本、用于SPR生物传感器的葡聚糖修饰金膜芯片的再生方法及其表征和应用。用此方法再生的芯片能被循环伏安法和原子力显微镜很好地表征,并成功地用于抗前列腺特异性抗原(prostate-specific antigen,PSA)固定和PSA检测, 同时测定了PSA与其抗体之间的动力学和亲和常数。     

Portable 24-analyte surface plasmon resonance instruments for rapid, versatile biodetection

Field use of surface plasmon resonance (SPR) biosensors for environmental and defense applications such as detection and identification of biological warfare agents has been hampered by lack of rugged, portable, high-performance instrumentation. To meet this need, we have developed compact multi-analyte SPR instruments based on Texas Instruments' Spreeta sensing chips. The instruments weigh 3 kg and are built into clamshell enclosures measuring 28 cm x 22 cm x 13 cm. Functions are divided between an electronics unit in the base of the box and a fluidics assembly in the lid. Automated valves and pumps implement an injection loop flow system that allows sensors to be exposed to sample, rinsed, and treated with additional reagents (such as secondary antibodies) under computer control. Injected samples flow over the surfaces of eight sensor chips fastened into a temperature-controlled silicone flowcell. Each chip has 3 sensing regions, for a total detection of 24 areas that can be simultaneously monitored by SPR. Coating these areas with appropriate antibodies or other receptors allows a sample to be screened for up to 24 different substances simultaneously. The instruments report refractive index (RI) values every second, with a typical noise level of 1-3 x 10(-6) RI units. The design of the device is described, and performance is illustrated with detection of six distinct analytes ranging from small molecules to whole microbes during the course of a single experiment.     

Detection of gastric carcinoma-associated antigen MG7-Ag in human sera using surface plasmon resonance sensor

Background: MG7-Ag is a kind of gastric cancer-specific tumor-associated antigen and has been investigated to serve as a marker of gastric cancer for early diagnosis. Methods: Surface plasmon resonance (SPR) sensor was used for the detection of MG7-Ag in the sera of gastric cancer patients to develop an innovative, simple and rapid assay method for early diagnosis. The specific monoclonal MG7 antibodies were used as capture and detection receptors which were immobilized on the surface of SPR sensor chips for MG7-Ag identification in the human sera. The measurements include 9 cases of gastric cancer patients and 2 cases of healthy blood donors and a MKN45 cancer cell lysate solution sample for positive control. Results: The binding of MG7-Ag onto the sensor surface was observed from SPR spectra. The sera of most gastric cancer patients revealed much higher expression level of MG7-Ag than healthy human sera did in SPR measurement. Conclusion: The initial results demonstrate that the SPR biosensor has the potential for its application in the early diagnosis of gastric cancer. However, more tests need to be done to confirm the detection limitation and the criterion for cancer risk evaluation in early diagnosis.     

A surface plasmon resonance approach to monitor toxin interactions with an isolated voltage-gated sodium channel paddle motif

Animal toxins that inhibit voltage-gated sodium (Na_v) channel fast inactivation can do so through an interaction with the S3b–S4 helix-turn-helix region, or paddle motif, located in the domain IV voltage sensor. Here, we used surface plasmon resonance (SPR), an optical approach that uses polarized light to measure the refractive index near a sensor surface to which a molecule of interest is attached, to analyze interactions between the isolated domain IV paddle and Na_v channel–selective α-scorpion toxins. Our SPR analyses showed that the domain IV paddle can be removed from the Na_v channel and immobilized on sensor chips, and suggest that the isolated motif remains susceptible to animal toxins that target the domain IV voltage sensor. As such, our results uncover the inherent pharmacological sensitivities of the isolated domain IV paddle motif, which may be exploited to develop a label-free SPR approach for discovering ligands that target this region.     

Phosphoinositide-containing polymerized liposomes: stable membrane-mimetic vesicles for protein-lipid binding analysis

Stable phosphoinositide (PIP(n))-containing liposomes were prepared using polydiacetylene photochemistry. Tethered pentacosadiynyl inositol polyphosphate (InsP(n)) analogues of Ins(1,3,4)P(3), Ins(1,4,5)P(3), and Ins(1,3,4,5)P(4) were synthesized, incorporated into vesicles made up of diyne-phosphatidylcholine and -phosphatidylethanolamine, and polymerized by UV irradiation. The polymerized liposome nanoparticles showed markedly increased stability over conventional PIP(n)-containing vesicles as a result of the covalent conjugated ene-yne network in the acyl chains. The polymerized liposomes were specifically recognized by PIP(n) binding PH domains in liposome overlay assays and amplified luminescent proximity homogeneous assays. Moreover, the biotin moiety allowed attachment of the nanoparticles to a streptavidin-coated sensor chips in surface plasmon resonance (SPR) sensor. The PIP(n) headgroups displayed on SPR sensors showed higher affinities for PH domains and PIP(n) monoclonal antibodies than did monomeric PIP(n)-analogues with biotinylated acyl chains.     

The Microtubule Plus-End Tracking Proteins SPR1 and EB1b Interact to Maintain Polar Cell Elongation and Directional Organ Growth in Arabidopsis

The microtubule plus-end tracking proteins (+TIPs) END BINDING1b (EB1b) and SPIRAL1 (SPR1) are required for normal cell expansion and organ growth. EB proteins are viewed as central regulators of +TIPs and cell polarity in animals; SPR1 homologs are specific to plants. To explore if EB1b and SPR1 fundamentally function together, we combined genetic, biochemical, and cell imaging approaches in Arabidopsis thaliana. We found that eb1b-2 spr1-6 double mutant roots exhibit substantially more severe polar expansion defects than either single mutant, undergoing right-looping growth and severe axial twisting instead of waving on tilted hard-agar surfaces. Protein interaction assays revealed that EB1b and SPR1 bind each other and tubulin heterodimers, which is suggestive of a microtubule loading mechanism. EB1b and SPR1 show antagonistic association with microtubules in vitro. Surprisingly, our combined analyses revealed that SPR1 can load onto microtubules and function independently of EB1 proteins, setting SPR1 apart from most studied +TIPs in animals and fungi. Moreover, we found that the severity of defects in microtubule dynamics in spr1 eb1b mutant hypocotyl cells correlated well with the severity of growth defects. These data indicate that SPR1 and EB1b have complex interactions as they load onto microtubule plus ends and direct polar cell expansion and organ growth in response to directional cues.     

Analysis of protein interactions on protein arrays by a wavelength interrogation-based surface plasmon resonance biosensor

We have investigated whether surface plasmon resonance (SPR) sensors based on the wavelength interrogation are able to analyze protein interactions on protein arrays. The spectral SPR sensor was self-constructed and its detection limit, expressed as the minimal refractive index variation, was calculated to be 6.6x10(-5) with the signal fluctuation of 1.0x10(-5). The protein array surface was modified by a mixed thiol monolayer to immobilize proteins. Protein arrays were analyzed by the line-scanning mode of the SPR sensor, which scanned every 100 microm along the central line of array spots and the scanned results were presented by color spectra from blue to red. Glutathione S-transferase (GST)-rac1 caused a concentration-dependent increase of SPR wavelength shift on protein arrays. The surface structure of the protein arrays was analyzed by atomic force microscopy. Specific interactions of antigens with antibodies were analyzed on the protein arrays by using three antibodies and eight proteins. These results suggest that the wavelength interrogation-based SPR sensor can be used as the biosensor for the high-throughput analysis of protein interactions on protein arrays.     

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)

Protein-protein interactions are pivotal to most, if not all, physiological processes, and understanding the nature of such interactions is a central step in biological research. Surface Plasmon Resonance (SPR) is a sensitive detection technique for label-free study of bio-molecular interactions in real time. In a typical SPR experiment, one component (usually a protein, termed ''ligand'') is immobilized onto a sensor chip surface, while the other (the ''analyte'') is free in solution and is injected over the surface. Association and dissociation of the analyte from the ligand are measured and plotted in real time on a graph called a sensogram, from which pre-equilibrium and equilibrium data is derived. Being label-free, consuming low amounts of material, and providing pre-equilibrium kinetic data, often makes SPR the method of choice when studying dynamics of protein interactions. However, one has to keep in mind that due to the method''s high sensitivity, the data obtained needs to be carefully analyzed, and supported by other biochemical methods. SPR is particularly suitable for studying membrane proteins since it consumes small amounts of purified material, and is compatible with lipids and detergents. This protocol describes an SPR experiment characterizing the kinetic properties of the interaction between a membrane protein (an ABC transporter) and a soluble protein (the transporter''s cognate substrate binding protein).     

A novel system for convenient detection of low-affinity receptor-ligand interactions: chelator-lipid liposomes engrafted with recombinant CD4 bind to cells expressing MHC class II

The interactions of cell surface receptors with their ligands, crucial for initiating many immunological responses, are often stabilized by receptor dimerization/oligomerization, and by multimeric interactions between receptors on one cell with their ligands or cognate receptors on the apposing cell. Current techniques for studying receptor-ligand interactions, however, do not always allow receptors to move laterally to enable dimerization/ oligomerization, or to interact multimerically with ligands on cell surfaces. For these reasons detection of low- affinity receptor-ligand interactions has been difficult. Utilizing a novel chelator-lipid, nitrilotriacetic acid di-tetradecylamine (NTA-DTDA), we have developed a convenient liposome system for directly detecting low-affinity receptor-ligand interactions. Our studies using recombinant soluble forms of murine CD40 and B7.1, and murine and human CD4, each possessing a hexhistidine tag, showed that these proteins can be anchored or 'engrafted' directly onto fluorescently labelled liposomes via a metal-chelating linkage with NTA-DTDA, permitting them to undergo dimerization/oligomerization and multimeric binding with ligands on cells. Fluorescence- activated cell sorter (FACS) analyses demonstrated that while there is little if any binding of soluble forms of murine CD40 and B7.1, and murine and human CD4 to cells, engrafted liposomes bind specifically to cells expressing the appropriate cognate receptor, often giving a fluorescence 4-6-fold above control cells. Such liposomes could detect directly the low-affinity interaction of murine CD40 and B7.1 with CD154- and CD28-expressing cells, respectively, and the interaction of CD4 with MHC Class II, which has hitherto defied direct detection except through mutational analysis and mAb blocking studies.     

Immobilisation of DNA probes for the development of SPR-based sensing

An immobilisation procedure based on the direct coupling of thiol-derivatised oligonucleotide probes to bare gold sensor surfaces has been used for DNA sensing applications. The instrumentation used relies on surface plasmon resonance (SPR) transduction; in particular the commercially available instruments BIACORE X and SPREETA, have been employed in this study. The performances of the SPR-based DNA sensors resulting from direct coupling of thiol-derivatised DNA probes onto gold chips, have been studied in terms of the main analytical parameters, i.e. selectivity, sensitivity, reproducibility, analysis time, etc. A comparison between the thiol-derivatised immobilisation approach and a reference immobilisation method, based on the coupling of biotinylated oligonucleotide probes onto a streptavidin coated dextran sensor surface, using synthetic complementary oligonucleotides has been discussed. Finally, a denaturation method to obtain ssDNA ready for hybridisation analysis has been applied to polymerase chain reaction (PCR) amplified samples, for the detection of genetically modified organisms (GMOs).