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。     

Real-time label-free immunoassay of interferon-gamma and prostate-specific antigen using a Fiber-Optic Localized Surface Plasmon Resonance sensor

A Fiber-Optic Localized Surface Plasmon Resonance (FO LSPR) sensor was fabricated using spherical gold nanoparticles (Au NPs) on a flattened end-face of the optical fiber. The Au NPs were easily synthesized by the Turkevich method and were immobilized on the end-face of the optical fiber by using a self-assembled monolayer (SAM). In order to examine the possibility of its application as a biosensor for label-free immunoassays, the fabricated FO LSPR sensor was used for the detection of the antibody-antigen reaction of interferon-gamma (IFN-γ) and the limit of detection (LOD) was approximately 2pg/ml. Herein, The antibodies and bovine serum albumins (BSAs) were immobilized on the Au NPs by physisorption. Also, the FO LSPR sensor was used for the detection of a prostate-specific antigen (PSA) and the LOD was 1pg/ml below. The fabricated FO LSPR sensor can be used for real-time label-free immunoassay having fast detection time, high resolution and sensitivity. In addition, the proposed sensor platform has the advantages of low cost, simple optical setup, remote sensing, simple fabrication, real-time detection, low sample volume, and potential application to in-vivo detection systems.     

A label-free optical technique for detecting small molecule interactions

A novel approach for the label-free detection of molecular interactions is presented in which a colorimetric resonant grating is used as a surface binding platform. The grating, when illuminated with white light, is designed to reflect only a single wavelength. When molecules are attached to the surface, the reflected wavelength (color) is shifted due to the change of the optical path of light that is coupled into the grating. By linking receptor molecules to the grating surface, complementary binding molecules can be detected without the use of any kind of fluorescent probe or radioactive label. The detection technique is capable of detecting the addition and removal of small molecules as they interact with receptor molecules on the sensor surface or enzymes in the solution surrounding the sensor. Two assays are presented to exemplify the detection of small molecule interactions with the biosensor. First, an avidin receptor layer is used to detect 244 Da biotin binding. Second, a protease assay is performed in which a 136 Da p-nitroanilide (pNA) moeity is cleaved from an immobilized substrate. Because the sensor structure can be embedded in the plastic surfaces of microtiter plates or the glass surfaces of microarray slides, it is expected that this technology will be most useful in applications where large numbers of biomolecular interactions are measured in parallel, particularly when molecular labels will alter or inhibit the functionality of the molecules under study. Screening of pharmaceutical compound libraries with protein targets, and microarray screening of protein-protein interactions for proteomics are examples of applications that require the sensitivity and throughput afforded by this approach.     

Online Monitoring of Solid-Phase Peptide Syntheses on Glass-Type Surfaces Using White Light Interference

Label-free optical detection methods are of particular value for the investigation of biomolecular interactions. A label-free method based on reflectometric interference spectroscopy is described which enables both the on-line monitoring of solid phase peptide syntheses and subsequently the determination of antibody binding to these peptides without cleavage from the support. The method uses SiO(2) transducers that were modified with diaminopolyethylene glycol. The stepwise coupling of different amino acids to the transducer surface was investigated and the complete monitoring of the synthesis of a viral epitope was performed. The success of the synthesis was proven via binding of a specific monoclonal antibody to the transducer-bound product. In order to demonstrate specificity the binding was inhibited with the same peptide epitope added in solution. The approach is attractive especially in the field of high throughput screening since both the synthesis and the interaction with the biological receptor can be monitored using the same technique.     

A streptavidin surface on planar glass substrates for the detection of biomolecular interaction

Based on the requirements of biomolecular interaction analysis on direct optical transducers, a streptavidin surface is examined. A general protocol was developed allowing the immobilization of biotinylated compounds using the rife biotin-streptavidin system. This type of surface modification can be applied to all biosensors using glass surfaces as sensor devices. Reflectometric interference spectroscopy (RIfS), a label-free, direct optical method was used to demonstrate the quality of the transducer surfaces. The surface modification is based on an aminofunctionalized polyethylene glycol layer covalently bound to the silica surface of the transducer and shows very little nonspecific binding. Biotin molecules can be easily coupled on such layers. Streptavidin followed by a biotinylated estrone derivative was immobilized by incubation of the biotinylated transducer surface. For the streptavidin layer we obtained interference signals corresponding to a protein monolayer. Finally, using a surface prepared as described above, biomolecular interaction experiments with an antibody against estrone were carried out to show the quality of the transducer surface. With RIfS all of the affinity-based surface modifications can be detected online and time resolved.     

Evanescent wave absorbance based fiber optic biosensor for label-free detection of E. coli at 280 nm wavelength

A novel label-free technique for the detection of pathogens based on evanescent wave absorbance (EWA) changes at 280 nm from a U-bent optical fiber sensor is demonstrated. Bending a decladded fiber into a U-shaped structure enhances the penetration depth of evanescent waves and hence sensitivity of the probe. We show that the enhanced EWA response from such U-bent probes, caused by the inherent optical absorbance properties of bacterial cells or biomolecules specifically bound to the sensor surface, can be exploited for the detection of pathogens. A portable optical set-up with a UV light emitting diode, a spectrometer and U-bent fiber optic probe of 200 μm core diameter, 0.75 mm bend radius and effective probe length of 1cm demonstrated an ability to detect less than 1000 cfu/ml.     

Epitope-mapping of transglutaminase with parallel label-free optical detection

The gastrointestinal disorder coeliac disease (CD) is induced by the ingestion of wheat gluten and is characterized by damage of the typical structure of the intestinal mucosa. The enzyme tissue transglutaminase (tTGase) was identified as the major target of disease-specific antibodies in-patients. We performed an epitope fine-mapping with a series of pentadecapeptides synthesized using parallel multiple peptide synthesis. For the detection of biomolecular interactions a label-free parallel method, reflectometric interference spectroscopy (RIfS), was used. This is the first optical label-free method adapted to a high throughput screening (HTS) format and the experimental results demonstrate its applicability as a biological screening device. A high titer of anti-tTGase antibodies is found in the serum of coeliac patients. We have taken the first step towards a fast non-surgical test for the detection of these antibodies. In order to identify and characterize a continuous epitope with high affinity against the anti-tTGase antibody a screening of 21 pentadecapeptides has been accomplished with the parallel RIfS system. A single channel RIfS-system with high resolution was used to determine binding constants of identified peptides with high affinity.     

Nanogap Dielectric Spectroscopy for Aptamer-Based Protein Detection

Among the various label-free methods for monitoring biomolecular interactions, capacitive sensors stand out due to their simple instrumentation and compatibility with multiplex formats. However, electrode polarization due to ion gradient formation and noise from solution conductance limited early dielectric spectroscopic measurements to high frequencies only, which in turn limited their sensitivity to biomolecular interactions, as the applied excitation signals were too fast for the charged macromolecules to respond. To minimize electrode polarization effects, capacitive sensors with 20 nm electrode separation were fabricated using silicon dioxide sacrificial layer techniques. The nanoscale separation of the capacitive electrodes in the sensor results in an enhanced overlapping of electrical double layers, and apparently a more ordered “ice-like” water structure. Such effects in turn reduce low frequency contributions from bulk sample resistance and from electrode polarization, and thus markedly enhance sensitivity toward biomolecular interactions. Using these nanogap capacitive sensors, highly sensitive, label-free aptamer-based detection of protein molecules is achieved.     

Real-time and Label-free Bio-sensing of Molecular Interactions by Surface Plasmon Resonance: A Laboratory Medicine Perspective

Radioactive, chromogenic, fluorescent and other labels have long provided the basis of detection systems for biomolecular interactions including immunoassays and receptor binding studies. However there has been unprecedented growth in a number of powerful label free biosensor technologies over the last decade. While largely at the proof-of-concept stage in terms of clinical applications, the development of more accessible platforms may see surface plasmon resonance (SPR) emerge as one of the most powerful optical detection platforms for the real-time monitoring of biomolecular interactions in a label-free environment.In this review, we provide an overview of SPR principles and current and future capabilities in a diagnostic context, including its application for monitoring a wide range of molecular markers of disease. The advantages and pitfalls of using SPR to study biomolecular interactions are discussed, with particular emphasis on its potential to differentiate subspecies of analytes and the inherent ability for quantitation through calibration-free concentration analysis (CFCA). In addition, recent advances in multiplex applications, high throughput arrays, miniaturisation, and enhancements using noble metal nanoparticles that promise unprecedented sensitivity to the level of single molecule detection, are discussed.In summary, while SPR is not a new technique, technological advances may see SPR quickly emerge as a highly powerful technology, enabling rapid and routine analysis of molecular interactions for a diverse range of targets, including those with clinical applicability. As the technology produces data quickly, in real-time and in a label-free environment, it may well have a significant presence in future developments in lab-on-a-chip technologies including point-of-care devices and personalised medicine.     

A real-time PCR-based method for determining the surface coverage of thiol-capped oligonucleotides bound onto gold nanoparticles

Here we report a real-time PCR-based method for determining the surface coverage of dithiol-capped oligonucleotides bound onto gold nanoparticles alone and in tandem with antibody. The detection of gold nanoparticle-bound DNA is accomplished by targeting the oligonucleotide with primer and probe binding sites, amplification of the oligonucleotide by PCR, and real-time measurement of the fluorescence emitted during the reaction. This method offers a wide dynamic range and is not dependant on the dissociation of the oligonucleotide strands from the gold nanoparticle surface; the fluorophore is not highly quenched by the gold nanoparticles in solution during fluorescence measurements. We show that this method and a fluorescence-based method give equivalent results for determining the surface coverage of oligonucleotides bound onto 13 or 30 nm gold nanoparticles alone and in tandem with antibody. Quantifying the surface coverage of immobilized oligonucleotides on metallic nanoparticle surfaces is important for optimizing the sensitivity of gold nanoparticle-based detection methods and for better understanding the interactions between thiol-functionalized oligonucleotides and gold nanoparticles.     

Direct immobilization of gangliosides onto gold-carboxymethyldextran sensor surfaces by hydrophobic interaction: applications to antibody characterization

We describe a novel immobilization technique to investigate interactions between immobilized gangliosides (GD3, GM1, and GM2) and their respective antibodies, antibody fragments, or binding partners using an optical biosensor. Immobilization was performed by direct injection onto a carboxymethyldextran sensor chip and did not require derivatization of the sensor surface or the ganglioside. The ganglioside appeared to bind to the sensor surface by hydrophobic interaction, leaving the carbohydrate epitope available for antibody or, in the case of GM1, cholera toxin binding. The carboxyl group of the dextran chains on the sensor surface did not appear to be involved in the immobilization as evidenced by equivalent levels of immobilization following conversion of the carboxyl groups into acyl amino esters, but rather the dextran layer provided a hydrophilic coverage of the sensor chip which was essential to prevent nonspecific binding. This technique gave better reactivity and specificity for anti- ganglioside monoclonal antibodies (anti-GD3: KM871, KM641, R24; and anti-GM2: KM966) than immobilization by hydrophobic interaction onto a gold sensor surface or photoactivated cross-linking onto carboxymethydextran. This rapid immobilization procedure has facilitated detailed kinetic analysis of ganglioside/antibody interactions, with the surface remaining viable for a large number of cycles (>125). Kinetic constants were determined from the biosensor data using linear regression, nonlinear least squares and equilibrium analysis. The values of kd, ka, and KAobtained by nonlinear analysis (KAKM871 = 1.05, KM641 = 1.66, R24 = 0.14, and KM966 = 0.65 x 10(7) M- 1) were essentially independent of concentration and showed good agreement with data obtained by other analytical methods.      

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。     

Long period grating based biosensor for the detection of Escherichia coli bacteria

In this paper we report a stable, label-free, bacteriophage-based detection of Escherichia coli (E. coli) using ultra sensitive long-period fiber gratings (LPFGs). Bacteriophage T4 was covalently immobilized on optical fiber surface and the E. coli binding was investigated using the highly accurate spectral interrogation mechanism. In contrast to the widely used surface plasmon resonance (SPR) based sensors, no moving part or metal deposition is required in our sensor, making the present sensor extremely accurate, very compact and cost effective. We demonstrated that our detection mechanism is capable of reliable detection of E. coli concentrations as low as 10(3)cfu/ml with an experimental accuracy greater than 99%.     

Real-time analysis of antibody binding interactions with immobilized E. coli O157:H7 cells using the BIAcore

The kinetic properties of antibody-antigen reaction and other interacting macromolecules can be analyzed in real-time using the surface plasmon resonance biosensor (BIAcore). The interactions of an antibody against Escherischia coli O157:H7 were studied using immobilized whole cells. The bacterial sensor surface was evaluated with anti-E. coli in a continuous flow system. Regeneration of the sensor surface with guanidine-HCl was more effective than with phosphoric acid and resulted in better binding reproducibility. The determined kinetic values, association and dissociation rate constants, can be used in the development of rapid immuno-techniques. This study also provides the basis to evaluate real-time interactions of macromolecules with immobilized cells.     

Quantification of antibody production of individual hybridoma cells by surface plasmon resonance imaging

Surface plasmon resonance imaging (SPRi) is most frequently used for the label-free measurement of biomolecular interactions. Here we explore the potential of SPRi to measure antibody production of individual hybridoma cells. As a model system, cells from a hybridoma, producing monoclonal antibodies recognizing epithelial cell adhesion molecule (EpCAM), were used. Recombinant human EpCAM protein was immobilized on an SPR sensor and hybridoma cells were introduced into an IBIS MX96 SPR imager and the SPRi response was followed for 10 h. SPRi responses were detected on the spots of the sensor only where ligands of the produced antibody were present. By measuring the SPRi signals on individual cells the antibody production of the individual cells was measured and production rates were calculated. For 53 single EpCAM hybridoma cells the production ranged from 0.16 to 11.95 pg (mean 2.96 pg per cell, SD 2.51) over a period of 10 h. Antibody excretion per cell per hour ranged from 0.02 to 1.19 pg (mean 0.30, SD 0.25). Here we demonstrate for the first time that antibody production of individual cells can be measured and quantified by SPRi, opening a new avenue for measuring excretion products of individual cells.     

Real time analysis of intact organelles using surface plasmon resonance

Membrane proteins remain refractory to standard protein chip analysis. They are typically expressed at low densities in distinct subcellular compartments, their biological activity can depend on assembly into macromolecular complexes in a specific lipid environment. We report here a real-time, label-free method to analyze membrane proteins inserted in isolated native synaptic vesicles. Using surface plasmon resonance-based biomolecular interaction analysis (Biacore), organelle capture from minute quantities of 10,000 g brain supernatant (1-10 microg) was monitored. Immunological and morphological characterization indicated that pure intact synaptic vesicles were immobilized on sensor chips. Vesicle chips were stable for days, allowing repetitive use with multiple analytes. This method provides an efficient way in which to characterize organelle membrane components in their native context. Organelle chips allow a broad range of measurements, including interactions of exogenous ligands with the organelle surface (kinetics, Kd), and protein profiling.     

In vitro binding interactions of oral bacteria with immobilized fructosyltransferase

AIMS: The objective of the present study was to explore the role of immobilized fructosyltransferase (FTF) in adhesion process. METHODS AND RESULTS: We investigated real-time biospecific interactions between several types of oral bacteria and recombinant FTF immobilized on a biosensor chip, using surface plasmon resonance technology. Streptococcus mutans, Streptococcus sobrinus and Actinomyces viscosus demonstrated significant binding to FTF. Actinomyces viscosus had a greater binding to FTF, with 373 Resonance Units (RU), than the other tested bacteria. The binding level to FTF of Strep. sobrinus was 320 RU, whereas Strep. mutans and Streptococcus salivarious show binding of 296 and 245 RU, respectively. The binding sensograms displayed different profiles for the tested bacteria at various cell density, suggesting a different affinity to immobilized FTF. CONCLUSIONS: The results from this study suggest that FTF may influence bacterial adherence and colonization of the dental biofilm. SIGNIFICANCE AND IMPACT OF THE STUDY: The biomolecular interaction analysis enables real-time monitoring of the interaction between adhesions of intact bacteria and their ligands, which might be crucial in the initial phase of biofilm development in vivo.     

Simultaneous determination of kinetic parameters for the binding of cholera toxin to immobilized sialic acid and monoclonal antibody using an array biosensor

Interactions between protein toxins and carbohydrate receptors are often semi-selective processes and the kinetic parameters that define the binding of a receptor to different toxins may vary with each interaction. In this study, we have determined the affinity constants for binding of cholera toxin (CT) to immobilized sialic acid and to anti-CT antibody (as a simultaneous reference) by measuring real-time binding processes using an array biosensor. N-Acetylneuraminic acid (Neu5Ac), a member of the sialic acid family, was covalently immobilized onto maleimide-activated planar waveguides via a thiol-terminated linker attached to the anomeric carbon of the sugar. Control antibodies were immobilized using two different approaches: covalent attachment onto maleimide-activated slides via the thiol on cysteine residues and non-covalent attachment using a biotin-NeutrAvidin linkage. Cy5-labeled CT was flowed over the immobilized receptors and the fluorescent intensity of the bound CT-receptor complex was recorded as a function of time. The association constants for CT binding to covalently attached Neu5Ac, to covalently attached anti-CT monoclonal antibody, and to antibody tethered by biotin-NeutrAvidin interactions were determined to be 1.3 x 10(8), 2.1 x 10(8) and 5.7 x 10(8)M(-1), respectively.     

Detection of CD133-marked cancer stem cells by surface plasmon resonance: Its application in leukemia patients

Here, we reported the development of a label-free and real-time surface plasmon resonance (SPR) based biosensor for cancer stem cells (CSCs) detection using cell surface biomarker; CD133. The fabricated biosensor was used for detection of this marker in some acute myeloid leukemia (AML) patients and the results were compared with those obtained from flow cytometry (FC) method. CD133 antibody was immobilized on the gold chip surface via EDC/NHS coupling method and binding of the candidate cells to the modified gold sensor surface was monitored after isolation of mononuclear cells from bone marrow of the patients. The method was validated in terms of various parameters such as CD133- antibody concentration and cell density. The CD133-marked cells were investigated in seven AML patients. All SPR results were compared with those obtained from FC method. A very good correlation (R² = 0.96) was obtained between SPR and FC responses related to CD133-marked cells densities. In conclusion, in this study, a label-free and real-time SPR cytometry method was developed to detect CD133 and it was successfully applied to follow this cancer stem cell biomarker in AML patients.     

Novel approach to analyzing RNA aptamer-protein interactions: toward further applications of aptamers

Surface plasmon-resonance analysis using a Biacore biosensor is a powerful tool for the detailed study of biomolecular interactions. The authors examined the methods of immobilizing proteins on the surface of NTA, SA, and CM5 sensor chips to study RNA aptamer-protein interactions. RNA aptamers and their deletion variants were loaded onto a protein-immobilized sensor chip, and their binding affinities were analyzed. Immobilizing the protein on a CM5 sensor chip via an anti-His-tag antibody was the only strategy that clearly detected the kinetic parameters of the interactions. DeltaNEO-III-14U, one of the deletion variants of the NS3 aptamer, had the highest binding affinity for the deltaNS3 protein in this study (KD = 4 x 10(-8)). Moreover, the 29-amino-acid spacer fragment was essential for protein immobilization using this strategy. This novel method will be useful in comparing the affinity of various RNA aptamers and selecting the most suitable candidates for a given target, as well as facilitating the in vitro selection procedure itself.