Detection of Escherichia coli 0157:H7 using a surface plasmon resonance biosensor

The BIAcore biosensor was used to detect binding of Escherichia coli O157:H7 with specific antibodies. Immobilized Protein A or Protein G captured antibodies which in turn bound to the bacteria. Alternatively, immobilized antibody captured the E. coli O157:H7 and the bacteria were further probed by a second antibody which enhanced the signal. The regenerated sensor surfaces were used for at least 50 separate analyses. The surface plasmon resonance biosensor has potential for use in rapid, real-time detection and identification of bacteria, and to study the interaction of organisms with different antisera or other molecular species. © Rapid Science Ltd. 1998     

Binding interactons of collagen I, laminin and fibronectin with immobilized Escherichia coli 0157:H7 using a surface plasmon resonance biosensor

Real-time interactions of collagen I, fibronectin, laminin, hyaluronic acid and chondroitin sulfate with immobilized Escherichia coli O157:H7 cells were studied with a surface plasmon resonance biosensor. Results showed that collagen I and laminin bound to the E.coli surface but fibronectin had very low binding while hyaluronic acid and chondroitin sulfate had no detectable interaction. Calcium ion inhibited laminin binding but enhanced collagen I binding. This research provides a model system to study the interactions of bacterial cells with extracellular matrix components. © Rapid Science Ltd. 1998     

Characterization of Ca and phosphocholine interactions with C-reactive protein using a surface plasmon resonance biosensor

The interactions between Ca²⁺ and C-reactive protein (CRP) have been characterized using a surface plasmon resonance (SPR) biosensor. The protein was immobilized on a sensor chip, and increasing concentrations of Ca²⁺ or phosphocholine were injected. Binding of Ca²⁺ induced a 10-fold higher signal than expected from the molecular weight of Ca²⁺. It was interpreted to result from the conformational change that occurs on binding of Ca²⁺. Two sites with different characteristics were distinguished: a high-affinity site with K_D = 0.03 mM and a low-affinity site with K_D = 5.45 mM. The pH dependencies of the two Ca²⁺ interactions were different and enabled the assignment of the different sites in the three-dimensional structure of CRP. There was no evidence for cooperativity in the phosphocholine interaction, which had K_D = 5 μM at 10 mM Ca²⁺. SPR biosensors can clearly detect and quantify the binding of very small molecules or ions to immobilized proteins despite the theoretically very low signals expected on binding, provided that significant conformational changes are involved. Both the interactions and the conformational changes can be characterized. The data have important implications for the understanding of the function of CRP and suggest that Ca²⁺ is an efficient regulator under physiological conditions.     

Poly-(3-hexylthiophene) self-assembled monolayer based cholesterol biosensor using surface plasmon resonance technique

Cholesterol oxidase (ChOx) has been covalently immobilized onto 1-fluoro-2-nitro-4-azidobenzene (FNAB) modified poly-(3-hexylthiophene) (P3HT) self-assembled monolayer (SAM) onto gold coated glass plates. These ChOx/FNAB/P3HT/Au bio-electrodes have been characterized using contact angle (CA) measurements, UV-vis spectroscopy, electrochemical impedance technique, cyclic voltammetric technique and atomic force microscopic (AFM) technique, respectively. The ChOx/FNAB/P3HT/Au bio-electrodes were utilized for the estimation of cholesterol concentration in standard solutions using surface plasmon resonance (SPR) technique. It is shown that this SPR biosensor has linearity from 50 to 500 mg/dl of cholesterol in solution with detection limit of 50 mg/dl, sensitivity of 1.0 4 m degrees /(mg dl), reusability of around 15 times and a shelf-life of about 10 weeks when stored at 4 degrees C.     

Advances in surface plasmon resonance biosensor analysis

The number and diversity of surface plasmon resonance (SPR) biosensor applications continue to increase. Evolutions in instrument and sensor chip technology, experimental methodology, and data analysis are making it possible to examine a wider variety of biomolecular interactions in greater mechanistic detail. SPR biosensors are poised to make a significant impact in basic research and pharmaceutical discovery.     

Label-free and high-sensitive detection of Salmonella using a surface plasmon resonance DNA-based biosensor

A method based on surface plasmon resonance (SPR) DNA biosensor has been developed for label-free and high-sensitive detection of Salmonella. A biotinylated single-stranded oligonucleotide probe was designed to target a specific sequence in the invA gene of Salmonella and then immobilized onto a streptavidin coated dextran sensor surface. The invA gene was isolated from bacterial cultures and amplified using a modified semi-nested asymmetric polymerase chain reaction (PCR) technique. In order to investigate the hybridization detection, experiments with different concentration of synthetic target DNA sequences have been performed. The calibration curve of synthetic target DNA had good linearity from 5 nM to 1000 nM with a detection limit of 0.5 nM. The proposed method was applied successfully to the detection of single-stranded invA amplicons from three serovars of Salmonella, i.e., Typhimurium, Enterica and Derby, and the responses to PCR products were related to different S. typhimurium concentrations in the range from 10(2) to 10(10) CFU mL(-1). While with this system to detect E. coli and S. aureus, no significant signal was observed, demonstrating good selectivity of the method. In addition, the hybridization can be completed within 15 min, and the excellent sensor surface regeneration allows at least 300 assay cycles without obvious loss of performance.     

Detection of TP53 mutation using a portable surface plasmon resonance DNA-based biosensor

A DNA-based surface plasmon resonance (SPR) biosensor has been developed for the detection of TP53 mutation using the inexpensive and commercially available instrument, SPREETA SPR-EVM-BT, from Texas Instruments. A direct immobilisation procedure, based on the coupling of thiol-derivatised oligonucleotide probes (Probe-C6-SH) to bare gold sensor surfaces, was optimized using synthetic oligonucleotides. Hybridisation reactions between the immobilised probe and a short sequence (26 mer) complementary, non-complementary and one-point mutation DNA were then investigated. The main analytical parameters of the sensor system were studied in detail including selectivity, sensitivity, reproducibility and analysis time. Finally, the sensor system was successfully applied to polymerase chain reaction (PCR)-amplified real samples, DNA extracted from both normal, wild-type, (Jurkat) and mutated (Molt 4), carrying the mutation at codon 248 of the TP53 cell lines. The results obtained demonstrate that the DNA-based SPR biosensor was able to distinguish sequences present in the various samples that differ only by one base; and hence, it appears to be a strong candidate technique for the detection of gene mutation.     

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.     

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.     

Kinetic studies of RNA-protein interactions using surface plasmon resonance

Although structural, biochemical, and genetic studies have provided much insight into the determinants of specificity and affinity of proteins for RNA, little is currently known about the kinetics that underlie RNA-protein interactions. Protein-RNA complexes are dynamic, and the kinetics of binding and release could influence many processes, such as the ability of RNA-binding proteins to compete for binding sites, the sequential assembly of ribonucleoprotein complexes, and the ability of bound RNA to move between cellular compartments. Therefore, to attain a complete and biologically relevant understanding of RNA-protein interactions, complex formation must be studied not only in equilibrated reactions, but also as a dynamic process. BIACORE, a surface plasmon resonance-based biosensor technology, allows intermolecular interactions to be measured in real time, and can provide both equilibrium and kinetic information about complex formation. This technology is a powerful tool with which to study the dynamics of RNA-protein interactions. We have used BIACORE extensively to obtain detailed insight into the interaction between RNA and proteins carrying RNA recognition motif domains. Here we discuss the physical principles on which BIACORE is based, and the required instrumentation. We describe how to design well-controlled RNA-protein interaction experiments aimed at yielding high-quality data, and outline the steps required for data analysis. In addition, we present examples to illustrate how kinetic studies have provided us with unique insights into the interaction of the spliceosomal U1A protein and the neuronal HuD protein with their respective RNA targets.     

Quantitative assay of hepatitis B surface antigen by using surface plasmon resonance biosensor

We performed a basic experiment for the rapid, on-line, real-time measurement of hepatitis B surface antigen using a surface plasmon resonance biosensor. We immobilized anti-HBsAg (hepatitis B surface antigen) polyclonal antibody, as a ligand, to the dextran layer on a CM5 chip surface that had previously been activated byN-hydroxysuccinimide. A sample solution containing HBsAg was fed through a microfluidic channel, and the reflecting angle change due to the mass increase from the binding was detected. The binding characteristics between HBsAg and its polyclonal antibody followed the typical monolayer adsorption isotherm. When the entire immobilized antibody had interacted, no additional, non-specific binding occurred, suggesting the immunoreaction was very specific. The bound antigen per unit mass of the antibody was independent of the immobilized ligand density. No significant steric hindrance was observed at an immobilization density of approximately 17.6 ng/mm². The relationship between the HBsAg concentration in the sample solution and the antigen bound to the ligand was linear up to ca. 40 μg/mL. This linearity was much higher than that of the ELISA method. It appeared the antigen-antibody binding increased as the immobilized ligand density increased. In summary, this study showed the potential of this SPR biosensor-based method as a rapid, simple and multi-sample on-line assay. Once properly validated, it may serve as a more efficient method for HBsAg quantification for replacing the ELISA.     

Interaction between collagens and glycosaminoglycans investigated using a surface plasmon resonance biosensor.

The interactions of glycosaminoglycans with collagens and other glycoproteins in extracellular matrix play important roles in cell adhesion and extracellular matrix assembly. In order to clarify the chemical bases for these interactions, glycosaminoglycan solutions were injected onto sensor surfaces on which collagens, fibronectin, laminin, and vitronectin were immobilized. Heparin bound to type V collagen, type IX collagen, fibronectin, laminin, and vitronectin; and chondroitin sulfate E bound to type II, type V, and type VII collagen. Heparin showed a higher affinity for type IX collagen than for type V collagen. On the other hand, chondroitin sulfate E showed the highest affinity for type V collagen. The binding of chondroitin sulfate E to type V collagen showed higher affinity than that of heparin to type V collagen. These data suggest that a novel characteristic sequence included in chondroitin sulfate E is involved in binding to type V collagen.     

Estrogen receptor binding assay of chemicals with a surface plasmon resonance biosensor

We have developed a simple assay method for the evaluation of estrogen receptor (ER) binding capacity of chemicals without the use of radio- or fluorescence-labeled compounds. We used the solution competition assay by the BIACORE biosensor, a surface plasmon resonance biosensor, with estradiol as a ligand, human recombinant ER(alpha) (hrER(alpha)) as a high molecular weight (hmw) interactant and test chemicals as analytes. For the ligand, aminated estradiol with a spacer molecule (E2-17PeNH) was synthesized and immobilized on a carboxymethyl dextran-coated sensor chip by the amine coupling method. The injection of the hmw interactant hrER(alpha) to the biosensor raised the sensorgram, indicating its binding to the ligand E2-17PeNH. The binding of test chemicals to hrERalpha was determined as a reduction in the hrER(alpha) binding to E2-17PeNH. The dissociation constant for the binding to hrER(alpha) was calculated for estrone (4.29 x 10(-9)M), estradiol (4.04 x 10(-10)M), estriol (8.35 x 10(-10)M), tamoxifen (2.16 x 10(-8)M), diethylstilbestrol (1.46 x 10(-10)M), bisphenol A (1.35 x 10(-6)M) and 4-nonylphenol (7.49 x 10(-6)M), by plotting the data according to an equation based on mass action law. This method can also be used as a high throughput screening method.     

Sensitivity enhancement of surface plasmon resonance biosensor with colloidal gold

We enhanced the sensitivity of surface plasmon resonance biosensor by the conversion of the real-time direct binding immunoassay into the sandwich immunoassay, in which colloidal gold particles coated with anti-mouse IgG was used. By the immobilization of anti-mouse IgG onto the carboxymethyl dextran surface of thin gold film, the direct binding of analyte (mouse IgG) onto the sensor chip, and the injection of colloidal gold particles coated with antimouse IgG, about 100 times of sensitivity enhancement was obtained. This result suggests that nanoparticles, which has a high refractive index, homogeneous ultrafine structure and capability of size control, would be applicable for the detection of very small quantity of biomaterial.     

Development of a biosensor microarray towards food screening, using imaging surface plasmon resonance

In this study we examined the possibilities of implementing direct and competitive immunoassay formats for small and large molecule detection on a microarray, using IBIS imaging surface plasmon resonance (iSPR) system. First, IBIS iSPR optics performance was evaluated. Using a glycerol calibration curve on underivatized surface we observed high baseline variability, but uniform and robust sensitivity between hundred regions of interest. Further on, a direct immunoassay for bovine IgG detection and a competitive immunoassay for gentamicin and neomycin were developed. The direct immunoassay for bovine IgG detection in a microarray format showed poor sensitivity in comparison to the assay performed in Biacore 3000, due to low immobilization efficiency on spots. The competitive immunoassay for parallel gentamicin and neomycin detection in a microarray format displayed sensitivity in the ngmL(-1) range, comparable with the sensitivity achieved in Biacore 3000 and in the range of maximum residue limits in milk, established in the European Union. We expect that, utilization of the IBIS iSPR system for food analysis, by screening high and low molecular weight compounds, will allow rapid and simultaneous detection of various ingredients and contaminants, providing the end-user with a detailed food profile. However, assay transfer from conventional SPR biosensors to the imaging microarray platform also presents new challenges, such as sufficient immobilization on spots, that must be addressed in future studies.     

Integrated-optic biosensor by electro-optically modulated surface plasmon resonance

We present a new integrated-optic surface plasmon resonance (SPR) biosensor based on electro-optical modulation. The SPR characteristics for the analyte concentration detection can be electro-optically modulated by applying the voltage on the electrodes of the biosensor fabricated on lithium niobate, which is an excellent electro-optic material. Two measurement methods, electro-optically modulated SPR spectral measurement and electro-optically modulated SPR intensity measurement, are demonstrated and their measurands are the SPR wavelength and the output optical intensity, respectively. Human serum albumin is coated on the gold film surface of the proposed biosensor to detect the concentration of beta-blocker, which is a remedy for heart disease. As the applied voltage increases such that the effective index of guided mode rises, the SPR wavelength shifts toward the long wavelength side and the output optical intensity at the wavelength of 632.8 nm diminishes. The linear regression slope of the relation between the measurand and the applied voltage is dependent on the analyte concentration and can be used to determine the concentration variation. Experimental results measured by the electro-optically modulated SPR methods are compared with those measured by the conventional spectral and intensity methods, and the effects of waveguide width on the biosensor performance are discussed.     

Survey of the 1999 surface plasmon resonance biosensor literature

The application of surface plasmon resonance biosensors in life sciences and pharmaceutical research continues to increase. This review provides a comprehensive list of the commercial 1999 SPR biosensor literature and highlights emerging applications that are of general interest to users of the technology. Given the variability in the quality of published biosensor data, we present some general guidelines to help increase confidence in the results reported from biosensor analyses.     

Analysis of human serum antibody-carbohydrate interaction using biosensor based on surface plasmon resonance

Surface plasmon resonance (SPR) was used to monitor the interaction of alphaGal-antibodies from human blood group O serum with linear blood group B-saccharides, employing Galalpha1-3Galbeta1-4GlcNAc-HSA immobilised on a sensor chip surface. Strong binding of antibodies, as evident from high relative response values exceeding 200 RU, was observed. The interaction was influenced by the nature of the oligosaccharide that was added to the antibody sample prior to measurement. For example, the addition of either of the linear B-saccharides Galalpha1-3Gal and Galalpha1-3Galbeta1-4GlcNAc produced complete inhibition of antibody binding to the sensor surface, whereas the addition of the related but non-specific blood group A saccharide, GalNAcalpha1-3(Fucalpha1-2)Gal, had little effect on binding. The technique was used for the rapid monitoring of the removal of alphaGal-antibodies from human serum by affinity columns, which contained either Galalpha1-3Gal or Galalpha1-3Galbeta1-4GlcNAc as ligand. The above carbohydrates are currently evaluated as inhibitors or as affinity ligands, in the prevention of hyperacute rejection during xenotransplantation.