Interaction between hammerhead ribozyme and RNA substrates measured by a surface plasmon resonance biosensor

Dynamic interactions between hammerhead ribozymes and RNA substrates were measured using the surface plasmon resonance (SPR) technology. Two in vitro transcribed substrates (non-cleavable and cleavable) were immobilised on streptavidin-coated dextran matrices and subsequently challenged with non-related yeast tRNA or two hammerhead ribozymes, both of which had previously been shown to exhibit functional binding and cleavage of complementary target RNAs. The target-binding domain of one of the ribozymes was fully complementary to a 16-ribonucleotide stretch on the immobilised substrates, while the other ribozyme had a nine-ribonucleotide complementarity. The two ribozymes could readily be differentiated with regard to affinity. Cleavage could be measured, using the ribozyme with full target complementarity to the cleavable substrate. In contrast, the ribozyme with lower affinity lacked cleavage activity. We suggest that SPR will be useful for investigations of ribozyme-substrate interactions.     

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.     

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.     

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.     

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.     

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.     

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     

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.     

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.     

Study of substrate-enzyme interaction between immobilized pyridoxamine and recombinant porcine pyridoxal kinase using surface plasmon resonance biosensor

Pyridoxal kinase (PK) is an important enzyme involved in bioactivation of vitamin B(6). Binding of PK with its substrate is the prerequisite step for the subsequent catalytic phosphorylation of the substrate. In the present study, a surface plasmon resonance biosensor (BIAcore) was employed to characterize the binding interaction between wild-type porcine PK and an immobilized substrate, pyridoxamine. Pyridoxamine was modified with 11-mercaptoundecanic acid and immobilized on a sensor chip through the formation of a self-assembled monolayer. The binding of PK to the immobilized pyridoxamine was followed in real time and the kinetic parameters were derived from non-linear analysis of the sensorgram. The effects of buffer pH, monovalent cations (Na(+), K(+)) and divalent cations (Mn(2+), Zn(2+), Mg(2+)) on the binding kinetics were determined. Optimal pH for PK-pyridoxamine interaction in the absence of divalent ions is at around 7.4. While K(+) increased and Na(+) decreased the binding affinity (K(A)) of PK to immobilized pyridoxamine, all divalent cations increased the K(A) of PK for pyridoxamine. Solution phase affinity measurement based on a competitive binding assay was used to determine the affinities of PK for different vitamin B(6) analogues. The order of affinity of PK for different analogues is: pyridoxal-oxime>pyridoxine>pyridoxamine>pyridoxal>pyridoxal phosphate. This is the first study to demonstrate that buffer conditions such as pH and concentration of monovalent and/or divalent ions can directly alter the binding of PK for its substrates. The quantitative kinetic and thermodynamic parameters obtained by SPR measurement provide the insight information into the catalytic activity of this enzyme.     

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.     

Detection of the toxin domoic acid from clam extracts using a portable surface plasmon resonance biosensor

The amnesic shellfish poison domoic acid is produced by marine algae of the genus Pseudo-nitzschia. We have developed a portable surface plasmon resonance (SPR) biosensor system for the detection of domoic acid. Because of concerns with domoic acid contamination of shellfish, there is a need for rapid field quantification of toxin levels in both shellfish and seawater. Antibodies were raised against domoic acid and affinity purified. These antibodies were used to develop competition- and displacement-based assays using a portable six-channel SPR system developed in our laboratories. Standard curves for detection of domoic acid in phosphate buffered saline and in diluted clam extracts analyzed by the competition-based SPR assay demonstrated a limit of detection of 3 ppb (10 nM) and a quantifiable range from 4 to 60 ppb (13–200 nM). Comparison of analyses for domoic acid levels in Pacific razor clams, Siliqua patula, containing moderate to high levels of domoic acid by the standard HPLC analysis protocol and the SPR-based assay gave an excellent correlation. This same technology should also function for detection of domoic acid in concentrated algal extracts or high dissolved levels in seawater.     

An enzyme-chromogenic surface plasmon resonance biosensor probe for hydrogen peroxide determination using a modified Trinder's reagent

An absorption-based surface plasmon resonance (SPR(Abs)) biosensor probe has been developed for simple and reproducible measurements of hydrogen peroxide using a modified Trinder's reagent (a chromogenic reagent). The reagent enabled the determination of the hydrogen peroxide concentration by the development of deep color dyes (lambda(max)=630nm) through the oxidative coupling reaction with N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline sodium salt monohydrate (MAOS; C(13)H(20)NNaO(4)S.H(2)O) and 4-aminoantipyrine (4-AA) in the presence of hydrogen peroxide and horseradish peroxidase (HRP). In the present study, urea as an adduct of hydrogen peroxide for color development could be omitted from the measurement solution. The measurement solution containing 5mM hydrogen peroxide was deeply colored at a high absorbance value calculated as 46.7cm(-1) and was directly applied to the SPR(Abs) biosensing without dilution. The measurement was simply performed by dropping the measurement solution onto the surface of the SPR sensor probe, and the SPR(Abs) biosensor response to hydrogen peroxide was obtained as a reflectivity change in the SPR spectrum. After investigation of the pH profiles in the SPR(Abs) biosensor probe, a linear calibration curve was obtained between 1.0 and 50mM hydrogen peroxide (r=0.991, six points, average of relative standard deviation; 0.152%, n=3) with a detection limit of 0.5mM. To examine the applicability of this SPR(Abs) biosensor probe, 20mM glucose detection using glucose oxidase was also confirmed without influence of the refractive index in the measurement solution. Thus, the SPR(Abs) biosensor probe employing the modified Trinder's reagent demonstrated applicability to other analyte biosensing tools.     

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.     

Monitoring a toxic bloom of Alexandrium minutum using novel microarray and multiplex surface plasmon resonance biosensor technology

Blooms of Alexandrium occur annually during the summer months in the North Channel of Cork Harbour on the south coast of Ireland. This study monitored an extensive bloom of the toxin producing Alexandrium minutum during the summer of 2011 with the use of the MIDTAL (Microarrays for the Detection of Toxic Algae) microarray and a prototype multiplex surface plasmon resonance (multi SPR) biosensor. Microarray signal intensities and toxin results from three testing platforms of the prototype multi SPR biosensor, commercial (CER) enzyme-linked immunosorbent assay (ELISA) and high performance liquid chromatography (HPLC) were compared against light microscopy counts. The main aim was to demonstrate the use of these methodologies to support national monitoring agencies by providing a faster and more accurate means of identifying and quantifying the harmful phytoplankton community and their toxins in natural water samples. Both the microarray signals and multi SPR biosensor results followed a significant trend with light microscopy results and both techniques indicated detection limits of <4000 cells of A. minutum in natural seawater samples.     

Binding interactions of Escherichia coli with globotetraosylceramide (globoside) using a surface plasmon resonance biosensor

Surface plasmon resonance with an alkane L1 chip was used to investigate the binding of uropathogenic Escherichia coli, carrying adhesion receptors, to globotetraosylceramide (globoside; GbO4). The immobilization of globoside was reproducible and resulted in a stable globoside layer on the L1 chip. The data indicated that the globoside-immobilized L1 chip could be used for studying interactions with live or chemically fixed E. coli. The results indicated that the dissociation time was significantly reduced in glutaraldehyde-fixed E. coli as compared to living cells. Overall, the report demonstrates the significance of the L1 chip in terms of sensitivity, specificity, handling, and speed when studying globoside/E. coli interactions. This model may assist in screening for compounds that can inhibit the binding of uropathogenic E. coli to glycolipid ligands on target cells.     

Analysis of the interaction between human interleukin-5 and the soluble domain of its receptor using a surface plasmon resonance biosensor

A surface plasmon resonance (SPR) biosensor was used to study the interaction of human interleukin-5 (hIL5) with its receptor. IL5 is a major growth factor in the production and activation of eosinophilis. The receptor for IL5 is composed of two subunits, α and β. The α subunit provides the specificity for IL5 and consist of an extracellular soluble domain, a single transmembrane region and a cytoplasmic tail. We expressed the soluble domain of the human IL5 receptor α subunit (shIL5Rα) and human IL5 (hIL5) in Drosophila. Both hIL5 and shIL5Rα were immobilized separately through amine groups onto the carboxylated dextran layer of sensor chips of the BIAcore? (Pharmacia) SPR biosensor after N-hydroxysuccinimide/carbodiimide activation of the chip surface. Interactions were measured for the complementary macromolecule, either shIL5Rα or hIL5, in solution. Kinetics of binding of soluble analyst to immobilized ligand were measured and from this the association rate constant, dissociation rate constant and equilibrium dissociation constant (K_d) were derived. With immobilized shIL5Rα and soluble hIL5, the measured K_d was 2 nM . A similar value was obtained by titration calorimetry. The K_d for Drosophila expressed receptor and IL5 is higher than the values reported for proteins expressed in different systems, likely due to differences in the methods of interaction analysis used for differences in protein glycosylation. Receptor-IL5 binding was relatively pH independent between pH 6.5 and 9.5. Outside this range the dissociation rate increased with compressibility little increased in association rate. The values obtained for the interaction of hIL5 and shIL5Rα were found to depend on which component was immobilized; the K_d was 5.5 nM with immobilized hIL5 and soluble shIL5Rα. The SPR biosensor provides a unified methodology to measure the interaction properties of shIL5Rα and hIL5 derivatives, mutants and mimetic as well as to evaluate potential antagonists of the receptor-cytokine interaction.     

A rapid biosensor chip assay for measuring of telomerase activity using surface plasmon resonance

Considerable interest has been focused on telomerase because of its potential use in assays for cancer diagnosis, and for anti-telomerase drugs as a strategy for cancer chemotherapy. A number of assays based on the polymerase chain reaction (PCR) have been developed for evaluation of telomerase activity. To overcome the disadvantages of the conventional telomerase assay [telomeric repeat amplification protocol (TRAP)] related to PCR artifacts and troublesome post-PCR procedures, we have developed a telomeric repeat elongation (TRE) assay which directly measures telomerase activity as the telomeric elongation rate by biosensor technology using surface plasmon resonance (SPR). 5′-Biotinylated oligomers containing telomeric repeats were immobilized on streptavidin-pretreated dextran sensor surfaces in situ using the BIACORE apparatus. Subsequently, the oligomers associated with the telomerase extracts were elongated in the BIACORE apparatus. The rate of TRE was calculated by measuring the SPR signals. We examined elongation rates by the TRE assay in 18 cancer and three normal human fibroblast cell lines, and 12 human primary carcinomas and matching normal tissues. The elongation rates increased in a concentration- and time-dependent manner. Those of cancer cells were two to 10 times higher than fibroblast cell lines and normal tissues. Telomerase activities and its inhibitory effects of anti-telomerase agents as measured by both the TRE and TRAP assays showed a good correlation. Our assay allows precise quantitative comparison of a wide range of human cells from somatic cells to carcinoma cells. TRE assay is suitable for practical use in the assessment of telomerase activity in preclinical and clinical trials of telomerase-based therapies, because of its reproducibility, rapidity and simplicity.