Real-time monitoring of odorant-induced cellular reactions using surface plasmon resonance

Surface plasmon resonance (SPR) is a powerful technique for measuring molecular interaction in real-time. SPR can be used to detect molecule to cell interactions as well as molecule to molecule interactions. In this study, the SPR-based biosensing technique was applied to real-time monitoring of odorant-induced cellular reactions. An olfactory receptor, OR I7, was fused with a rho-tag import sequence at the N-terminus of OR I7, and expressed on the surface of human embryonic kidney (HEK)-293 cells. These cells were then immobilized on a SPR sensor chip. The intensity of the SPR response was linearly dependent on the amount of injected odorant. Among all the aldehyde containing odorants tested, the SPR response was specifically high for octanal, which is the known cognate odorant for the OR I7. This SPR response is believed to have resulted from intracellular signaling triggered by the binding of odorant molecules to the olfactory receptors expressed on the cell surface. This SPR system combined with olfactory receptor-expressed cells provides a new olfactory biosensor system for selective and quantitative detection of volatile compounds.     

Aptamer–Au NPs conjugates-enhanced SPR sensing for the ultrasensitive sandwich immunoassay

The goal of this work is to explore the amplification effect of aptamer–gold nanoparticles (Au NPs) conjugates for ultrasensitive detection of large biomolecules by surface plasmon resonance (SPR). A novel sandwich immunoassay is designed to demonstrate the amplification effect of aptamer–Au NPs conjugates by using human immunoglobulin E (IgE) as model analyte. Human IgE, captured by immobilized goat anti-human IgE on SPR gold film, is sensitively detected by SPR spectroscopy with a lowest detection limit of 1 ng/ml after anti-human IgE aptamer–Au NPs conjugates is used as amplification reagent. Meanwhile, the non-specific adsorption of aptamer–Au NPs conjugates on goat anti-human IgE is confirmed by SPR spectroscopy and then it is minimized by treating aptamer–Au NPs conjugates with 6-mercaptohexan-1-ol (MCH). These results confirm that aptamer–Au NPs conjugates is a powerful sandwich element and an excellent amplification reagent for SPR-based sandwich immunoassay.     

A rapid serological assay for prediction of Salmonella infection status in slaughter pigs using surface plasmon resonance

We present a rapid surface plasmon resonance-based serological assay for the detection of Salmonella Typhimurium infection in pigs using the Plasmonic((R)) SPR device. Lipopolysaccharide (LPS, 10 microg mL(-1)) from Salmonella Typhimurium was immobilised by self-assembly on a hydrophobic SPR chip. Using this LPS-coated chip, it was possible to bind and detect the anti-Salmonella Typhimurium antibodies in serum of pigs infected with the bacteria. The developed SPR assay is able to differentiate between sera obtained from pigs having low, medium, and high levels of Salmonella infection. A commercial ELISA kit was used to classify the sera for levels of Salmonella infection on the basis of optical density (OD%). A strong positive correlation was observed between the SPR-based assay and the ELISA (n=38, r=0.90, p<0.01). The sensitivity and specificity of the assay are 0.93 and 0.87, respectively. The SPR-based assay is label-free and does not require any sample preparation or dilution steps. The total analysis time is 45 min for each serum sample. The assay was found to be specific for Salmonella Typhimurium and shows no cross-reactivity to Salmonella Choleraesuis or Escherichia coli antibodies. As no sample preparation is required the developed assay has the potential to be used as a reliable tool for Salmonella monitoring programmes in pork production.     

Label-free immunosensing for alpha-fetoprotein in human plasma using surface plasmon resonance

In this study, we attempted to develop a surface plasmon resonance (SPR)-based immunoassay sensor to detect alpha-fetoprotein (AFP) in human plasma at the nanogram level, as is required for clinical diagnosis of hepatocellular tumors. A self-assembled monolayer (SAM) surface of tri(ethylene glycol) (TEG) and carboxyl group-terminated hexa(ethylene glycol) (HEG) was employed to suppress the nonspecific adsorption of plasma components onto the sensor surface. AFP was detected by a sandwich-type immunoassay using two kinds of antibodies, primary and secondary, in this system. The SPR signal shift was further enhanced by applying an antibody (polyclonal) against the second antibody. With this method, the SPR signals were highly intensified, and so nanogram levels (ng/ml) of AFP could be easily detected with a high signal/noise ratio, as is necessary for clinical diagnosis. It is expected that our SPR-based immunoassay method can also be applicable to the detection of several other tumor markers that are present in low concentrations in human blood.     

Development of a surface plasmon resonance biosensor for real-time detection of osteogenic differentiation in live mesenchymal stem cells

Surface plasmon resonance (SPR) biosensors have been recognized as a useful tool and widely used for real-time dynamic analysis of molecular binding affinity because of its high sensitivity to the change of the refractive index of tested objects. The conventional methods in molecular biology to evaluate cell differentiation require cell lysis or fixation, which make investigation in live cells difficult. In addition, a certain amount of cells are needed in order to obtain adequate protein or messenger ribonucleic acid for various assays. To overcome this limitation, we developed a unique SPR-based biosensing apparatus for real-time detection of cell differentiation in live cells according to the differences of optical properties of the cell surface caused by specific antigen-antibody binding. In this study, we reported the application of this SPR-based system to evaluate the osteogenic differentiation of mesenchymal stem cells (MSCs). OB-cadherin expression, which is up-regulated during osteogenic differentiation, was targeted under our SPR system by conjugating antibodies against OB-cadherin on the surface of the object. A linear relationship between the duration of osteogenic induction and the difference in refractive angle shift with very high correlation coefficient was observed. To sum up, the SPR system and the protocol reported in this study can rapidly and accurately define osteogenic maturation of MSCs in a live cell and label-free manner with no need of cell breakage. This SPR biosensor will facilitate future advances in a vast array of fields in biomedical research and medical diagnosis.     

Surface plasmon resonance biosensor assay for the analysis of small-molecule inhibitor binding to human and parasitic phosphodiesterases

In the past decade, surface plasmon resonance (SPR) biosensor-based technology has been exploited more and more to characterize the interaction between drug targets and small-molecule modulators. Here, we report the successful application of SPR methodology for the analysis of small-molecule binding to two therapeutically relevant cAMP phosphodiesterases (PDEs), Trypanosoma brucei PDEB1 which is implicated in African sleeping sickness and human PDE4D which is implicated in a plethora of disease conditions including inflammatory pulmonary disorders such as asthma, chronic obstructive pulmonary disease and central nervous system (CNS) disorders. A protocol combining the use of directed capture using His-tagged PDE_CDs with covalent attachment to the SPR surface was developed. This methodology allows the determination of the binding kinetics of small-molecule PDE inhibitors and also allows testing their specificity for the two PDEs. The SPR-based assay could serve as a technology platform for the development of highly specific and high-affinity PDE inhibitors, accelerating drug discovery processes.     

Real-time assay method of lipid extraction activity

Intracellular lipid translocation is mediated by lipid transfer proteins and their functional impairments cause severe disorder in lipid metabolism. However, molecular mechanisms of protein-mediated lipid transfer remain unclear since conventional assay methods could not observe elementary processes in the lipid transfer reaction, such as lipid bilayer binding and lipid uptake. In this study, we found that ceramide extraction mediated by a ceramide trafficking protein (CERT) could be detected as decreasing the response of surface plasmon resonance (SPR). Based on this finding, we developed a novel real-time assay method that enables quantitative evaluation of the ceramide extraction activity of CERT, using the SPR technique. Performing this SPR-based assay using ceramide-embedded and ceramide-free lipid bilayers as ligands allows for the exclusive investigation of ceramide uptake processes, differentiating them from other CERT-membrane binding events. Furthermore, mutagenesis experiments of CERT using this SPR-based assay clearly elucidated whether an amino acid residue plays a role in the ceramide uptake process or the lipid bilayer binding process. This SPR-based assay method can separately evaluate the lipid extraction activity and lipid bilayer binding activity of the lipid transfer proteins, and provide more detailed information about lipid transfer phenomena.     

Detection of Intermolecular Interactions Based on Surface Plasmon Resonance Registration

Methods for registration of intermolecular interactions based on the phenomenon of surface plasmon resonance (SPR) have become one of the most efficient tools to solve fundamental and applied problems of analytical biochemistry. Nevertheless, capabilities of these methods are often insufficient to detect low concentrations of analytes or to screen large numbers of objects. That is why considerable efforts are directed at enhancing the sensitivity and efficiency of SPR-based measurements. This review describes the basic principles of the detection of intermolecular interactions using this method, provides a comparison of various types of SPR detectors, and classifies modern approaches to enhance sensitivity and efficiency of measurements.     

New trends in instrumental design for surface plasmon resonance-based biosensors

Surface plasmon resonance (SPR)-based biosensing is one of the most advanced label free, real time detection technologies. Numerous research groups with divergent scientific backgrounds have investigated the application of SPR biosensors and studied the fundamental aspects of surface plasmon polaritons that led to new, related instrumentation. As a result, this field continues to be at the forefront of evolving sensing technology. This review emphasizes the new developments in the field of SPR-related instrumentation including optical platforms, chips design, nanoscale approach and new materials. The current tendencies in SPR-based biosensing are identified and the future direction of SPR biosensor technology is broadly discussed.     

Specific Recognition of Biologically Active Amyloid-β Oligomers by a New Surface Plasmon Resonance-based Immunoassay and an in Vivo Assay in Caenorhabditis elegans

Soluble oligomers of the amyloid-β (Aβ) peptide play a key role in the pathogenesis of Alzheimer's disease, but their elusive nature makes their detection challenging. Here we describe a novel immunoassay based on surface plasmon resonance (SPR) that specifically recognizes biologically active Aβ oligomers. As a capturing agent, we immobilized on the sensor chip the monoclonal antibody 4G8, which targets a central hydrophobic region of Aβ. This SPR assay allows specific recognition of oligomeric intermediates that rapidly appear and disappear during the incubation of synthetic Aβ(1-42), discriminating them from monomers and higher order aggregates. The species recognized by SPR generate ionic currents in artificial lipid bilayers and inhibit the physiological pharyngeal contractions in Caenorhabditis elegans, a new method for testing the toxic potential of Aβ oligomers. With these assays we found that the formation of biologically relevant Aβ oligomers is inhibited by epigallocatechin gallate and increased by the A2V mutation, previously reported to induce early onset dementia. The SPR-based immunoassay provides new opportunities for detection of toxic Aβ oligomers in biological samples and could be adapted to study misfolding proteins in other neurodegenerative disorders.     

At-line quantification of bioactive antibody in bioreactor by surface plasmon resonance using epitope detection

We report an innovative at-line method to monitor concentration of bioactive antibody (i.e., antibody with conserved antigen-binding activity) secreted during bioreactor culture by the use of surface plasmon resonance (SPR)-based biosensor technology. In a first series of experiments, conditions for SPR-based measurements were validated off-line by monitoring bioactive antibody concentration in conditioned medium from 500-ml baffled flask hybridoma cell cultures. A fully automated experimental setup in which the SPR-based biosensor was harnessed to a bioreactor was then used at-line to monitor the concentration of bioactive antibody produced in a 3.5-L bioreactor. Quantitative SPR measurements performed both at-line and off-line were in excellent agreement with quantitative Western blotting followed by densitometry analyses. Thus, our experimental study confirms that SPR biosensors can be applied to at-line quantification of correctly folded proteins that are secreted by cells cultured in a bioreactor. Our experimental approach represents a novel and robust analytical strategy to be applied to the control and optimization of the production of bioactive secreted proteins.     

The fabrication of protein chip based on surface plasmon resonance for detection of pathogens

Protein chip based on surface plasmon resonance (SPR) was developed for detection of pathogens existing in contaminated environment, such as Escherichia coli O157:H7, Salmonella typhimurium, Legionella pneumophila, and Yersinia enterocolitica. Protein G was immobilized to endow the orientation of antibody molecules on the SPR surface. The pathogen binding of the protein chip was investigated by SPR spectroscopy. Consequently, it was found that the four kinds of pathogen could be selectively detected by using SPR-based protein chip.     

Design Analysis of Refractive Index Sensor with High Quality Factor Using Au-Al<Subscript>2</Subscript>O<Subscript>3</Subscript> Grating on Aluminum

We propose a surface plasmon resonance (SPR)-based refractive index sensor using gold-alumina grating over aluminum film for biosensing. Conventional SPR sensor based on gold grating exhibits broader SPR dips whereas that based on aluminum grating exhibits narrow reflection dip. A narrow reflection dip is desirable as it provides good resolution and improves the accuracy of measurement. Aluminum is less stable and generally is not preferred for an SPR-based sensor. It is more prone to being oxidized, which reduces the sensitivity and increases the width of the reflection dip of the sensor. While gold cannot provide narrow SPR reflection dips, but is used as an SPR active metal due to its more chemical stability. In order to improve the accuracy of gold grating-based sensor while taking care of oxidation problem of aluminum, in this paper, we propose a gold grating over aluminum film for SPR-based sensor and show that this configuration improves the sensitivity and the detection accuracy of the conventional sensor. Moreover, the oxidation problem is reduced to some extent as a part of aluminum is covered with gold. In order to completely avoid the oxidation of aluminum, we further propose to cover the exposed part of the aluminum with alumina and show that this configuration further improves the accuracy by reducing the width of the SPR reflection dip without affecting the sensitivity significantly. Numerical simulations show that sensitivity of proposed sensor is 270.33°/RIU with quality factor of more than 267.65 RIU^?1.     

A novel approach to protein expression profiling using antibody microarrays combined with surface plasmon resonance technology

We have previously described our systems for the high-throughput production of antibodies against mouse KIAA proteins and their validation (Proteomics 2004, 4, 1412-1416). Using our "libraries" of antibodies, we established a novel antibody microarray system in which surface plasmon resonance (SPR) technology is utilized for signal detection. Up to 400 real-time antibody-target bindings could be measured simultaneously within a single hour. This rapid detection was achieved by direct readout of the bindings using SPR technology. To evaluate our system, we assessed the reproducibility on crude protein samples and obtained satisfactorily reproducible results, exhibiting correlation values >0.92. Using this SPR-based antibody microarray system, we examined mKIAA protein expression in five different adult mouse tissues and identified the specific tissue expression patterns of several mKIAA proteins.     

Continuous flow immunosensor for highly selective and real-time detection of sub-ppb levels of 2-hydroxybiphenyl by using surface plasmon resonance imaging

A biosensor based on surface plasmon resonance (SPR) is developed for the detection of 2-hydroxybiphenyl (HBP). A monoclonal antibody against HBP (abbreviated hereafter as HBP-mAb) is developed and used for the detection of HBP by competitive SPR-based immunoassay and enzyme linked immunosorbent assay (ELISA) methods. A novel HBP-hapten compound, HBP-bovine serum albumin conjugate (HBP-BSA), derived by binding several HBP units with BSA by an aliphatic chain spacer is used in the development of antibody and for the functionalization of immunoprobes. HBP-BSA linked to the Au surface of the SPR sensor chip undergoes inhibitive immunoreaction with HBP-mAb in the presence of free HBP. The SPR-based immunoassay provides a rapid determination (response time: approximately 20 min) of the concentration of HBP in the range of 0.1-1000 ppb (ng/ml). Regeneration of the sensor chip is gained by treating the antibody-anchored SPR sensor chip with a pepsin solution (100 ppm (microg/ml); pH 2.0) for few minutes. The SPR sensor chip is reusable for the detection of HBP for more than 20 cycles with average loss of 0.35% reactivity per regeneration step. HBP concentration is determined as low as 0.1 and 3 ppb using the SPR sensor and ELISA measurements, respectively. The developed SPR sensor for HBP is free from interference by coexisting benzo[a]pyrene (BaP), 2,4-dichlorophenoxyacetic acid (2,4-D) and benz[a]anthracene; SPR angle shift obtained to the flow of HBP is almost same irrespective to the presence or absence of a same concentration of these carcinogenic polycyclic aromatic hydrocarbons together. The SPR sensor for HBP is proved to be applicable in simultaneous detection of HBP and BaP in parallel with another SPR sensor for BaP.     

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.     

Serum antibody screening by surface plasmon resonance using a natural glycan microarray

A surface plasmon resonance (SPR) based natural glycan microarray was developed for screening of interactions between glycans and carbohydrate-binding proteins (CBPs). The microarray contained 144 glycan samples and allowed the real-time and simultaneous screening for recognition by CBPs without the need of fluorescent labeling. Glycans were released from their natural source and coupled by reductive amination with the fluorescent labels 2-aminobenzamide (2AB) or anthranilic acid (AA) followed by high-performance liquid chromatography (HPLC) fractionation making use of the fluorescent tag. The released and labeled glycans, in addition to fluorescently labeled synthetic glycans and (neo)glycoproteins, were printed on an epoxide-activated chip at fmol amounts. This resulted in covalent immobilization, with the epoxide groups forming covalent bonds to the secondary amine groups present on the fluorescent glycoconjugates. The generated SPR glycan array presented a subset of the glycan repertoire of the human parasite Schistosoma mansoni. In order to demonstrate the usefulness of the array in the simultaneous detection of glycan-specific serum antibodies, the anti-glycan antibody profiles from sera of S. mansoni-infected individuals as well as from non-endemic uninfected controls were recorded. The SPR screening was sensitive for differences between infection sera and control sera, and revealed antibody titers and antibody classes (IgG or IgM). All SPR analyses were performed with a single SPR array chip, which required regeneration and blocking of the chip before the application of a serum sample. Our results indicate that SPR-based arrays constructed from glycans of natural or synthetic origin, pure or as mixture, can be used for determining serum antibody profiles as possible markers for the infection status of an individual.     

Optical surface plasmon resonance biosensors in molecular fishing

An optical biosensor employing surface plasmon resonance (SPR; SPR-biosensor) is a highly efficient instrument applicable for direct real time registration of molecular interactions without additional use of any labels or coupled processes. As an independent approach it is especially effective in analysis of various ligand receptor interactions. SPR-biosensors are used for validation of studies on intermolecular interactions in complex biological systems (affinity profiling of various groups of proteins, etc.). Recently, potential application of the SPR-biosensor for molecular fishing (direct affinity binding of target molecules from complex biological mixtures on the optical biosensor surface followed by their elution for identification by LCMS/MS) has been demonstrated. Using SPR-biosensors in such studies it is possible to solve the following tasks: (a) SPR-based selection of immobilization conditions required for the most effective affinity separation of a particular biological sample; (b) SPR-based molecular fishing for subsequent protein identification by mass spectrometry; (c) SPR-based validation of the interaction of identified proteins with immobilized ligand. This review considers practical application of the SPR technology in the context of recent studies performed in the Institute of Biomedical Chemistry on molecular fishing of real biological objects.     

Systematic Study of the Surface Plasmon Resonance Signals Generated by Cells for Sensors with Different Characteristic Lengths

The objectives of this study were to establish an in-depth understanding of the signals induced by mammalian cells in surface plasmon resonance (SPR) sensing. To this end, two plasmonic structures with different propagation and penetration distances were used: conventional surface plasmon resonance and long-range surface plasmon resonance. Long-range SPR showed a lesser sensitivity to the absolute number of round cells but a greater resolution due to its very narrow spectral dip. The effect of cell spreading was also investigated and the resonance angle of long-range SPR was mostly insensitive unlike in the conventional SPR counterpart. Experimental data was compared with suitable models used in the SPR literature. Although these simple averaging models could be used to describe some of the experimental data, important deviations were observed which could be related to the fact that they do not take into consideration critical parameters such as plasmon scattering losses, which is particularly crucial in the case of long-range SPR structures. The comparison between conventional and long-range SPR for cellular schemes revealed important fundamental differences in their responses to the presence of cells, opening new horizons for SPR-based cell assays. From this study, long-range SPR is expected to be more sensitive towards both the detection of intracellular events resulting from biological stimulation and the detection of microorganisms captured from complex biological samples.     

An indirect competitive immunoassay for insulin autoantibodies based on surface plasmon resonance

We have developed a sensitive and specific method based on surface plasmon resonance (SPR) for detection of insulin autoantibodies (IAA) in serum samples from individuals at high risk of developing type 1 diabetes (T1D). When measuring trace molecules in undiluted sera with label-free techniques like SPR, non-specific adsorption of matrix proteins to the sensor surface is often a problem, since it causes a signal that masks the analyte response. The developed method is an indirect competitive immunoassay designed to overcome these problems. Today, IAA is mainly measured in radio immunoassays (RIAs), which are time consuming and require radioactively labeled antigen. With our SPR-based immunoassay the overall assay time is reduced by a factor of >100 (4 days to 50min), while sensitivity is maintained at a level comparable to that offered by RIA.