Two-dimensional Surface Plasmon Resonance Research Articles

Two-dimensional material-enhanced surface plasmon resonance for antibiotic sensing

Two-dimensional (2D) materials attract attention from the academic community due to their excellent properties, and their wide application in sensing is expected to revolutionize environmental monitoring, medical diagnostics, and food safety. In this work, we systematically evaluate the effects of 2D materials on the Au chip surface plasmon resonance (SPR) sensor. The results reveal that 2D materials cannot improve the sensitivity of intensity-modulated SPR sensors. However, there exists an optimal real part of RI of 3.5–4.0 and optimal thickness when choosing nanomaterials for sensitivity enhancement of SPR sensors in angular modulation. In addition, the smaller the imaginary part of the nanomaterial RI, the higher the sensitivity of the proposed Au SPR sensor. The 2D material’s thickness needed for the highest sensitivity decreases with increasing real part and imaginary part of the RI. As a case study, we developed a 5 nm-thickness MoS2-enhanced SPR biosensor, which exhibited a low sulfonamides (SAs) detection limit of 0.05 μg/L based on a group-targeting indirect competitive immunoassay, nearly 12-fold lower than that of the bare Au SPR system. The proposed criteria help to shed light on the 2D material-Au surface interaction, which has greatly promoted the development of novel SPR biosensing with outstanding sensitivity.

IP-Dip-Based SPR Structure for Refractive Index Sensing of Liquid Analytes.

In this paper, we present a two-dimensional surface plasmon resonance structure for refractive index sensing of liquid analytes. The polymer structure was designed with a period of 500 nm and prepared in a novel IP-Dip polymer by direct laser writing lithography based on a mechanism of two-photon absorption. The sample with a set of prepared IP-Dip structures was coated by 40 nm thin gold layer. The sample was encapsulated into a prototyped chip with inlet and outlet. The sensing properties were investigated by angular measurement using the prepared solutions of isopropyl alcohol in deionized water of different concentrations. Sensitivity of 478–617 nm per refractive index unit was achieved in angular arrangement at external angle of incidence of 20°.

Evaluation of the Anti-Allergic Effect of Natural Medicines on Mast Cell by Using Two-Dimensional Surface Plasmon Resonance Observation

Evaluation of the Anti-Allergic Effect of Natural Medicines on Mast Cell by Using Two-Dimensional Surface Plasmon Resonance Observation

Continuous Monitoring of Differentiation Process of Neuronal Progenitor Cells By Two-Dimensional Surface Plasmon Resonance Observation at Single Cell Level

Introduction In our laboratory, we have examined to monitor intracellular events noninvasively by the two-dimensional surface plasmon resonance (2D-SPR) observation. In 2D-SPR observation, the refractive index change of the cell bottom can be observed as local changes in reflected light intensity. In previous study, we applied 2D-SPR observation to monitor the differentiation process of neuronal progenitor cells and found that the reflected light intensity at cell regions vibrated over time after neurite outgrowth. This finding suggested that it may be possible to evaluate neuronal differentiation very simply by monitoring of the SPR vibration phenomenon at the single cell region and analyzing the vibration frequency. However, in previous studies SPR observation before and after differentiation was done to the cells which were re-inoculated on a gold sensor chip for observation before one day. Therefore continuous observation of the differentiation process of the same progenitor cells have not been done yet. In this study, PC12 (Rat adrenal pheochromocytoma) as a neuronal progenitor cell model were cultured on a gold chip, and continual 2D-SPR observation of same cell regions was examined in differentiation process into neuron-like cells by NGF administration.Experimental method 400 μl of cell suspension of PC12 cells in DMEM medium containing 5% FBS and 10% HS was seeded in a sensor chamber in which PDMS well was adhered on a gold chip, and incubated overnight in an CO2 incubator. The next day, the medium was removed and replaced with a DMEM medium (containing 1% HS and 100 ng / ml NGF). At this time, the gas permeable membrane sheet was attached over the cell culture chamber. The gold chip chamber in which the cells were cultured was observed every day until the day 4 with a high resolution 2D-SPR instrument (model 2D-SPR04B, NTT-AT Japan). The day of the medium changed was set on the day 0. The sensor chamber was maintained in the incubator except 2D-SPR observation. A phase-contrast microscope was used to observe how PC12 cells adhered to the gold chip and differentiated into neuron-like cells by NGF administration and following neurite extension. At the same time, 2D-SPR observation of same PC12 cell regions on a gold chip was done to measure resonance angle and to monitor the reflected light intensity of the same cell region over time.Result and discussion Fig. 1 shows the time-course of the reflection light intensity at several cell regions on day 1 and day 2. By comparing the SPR signal on day1 and day2 it was demonstrated that the SPR signal at the cell region on day 2 showed significant vibration. The neurite outgrowth in PC12 cells on the day 2 was observed clearly. It is well known that polymerization and depolymerization of tubulin and actin filament, which are cytoskeletons, are actively performed for neurite out growth. Therefore we speculated that the SPR signal vibration of the reflection intensity might be due to the refractive index change by polymerization and depolymerization of the cytoskeleton closed to the cell bottom. In the presentation, we also mention the vibration frequency which way play a marker for differentiation evaluation, by the Fourier analysis of this vibration phenomenon. At the present time, 2D-SPR could not be observed after day 3 because the cell adhesion was weakened and cells were detached from the gold chip. We are now trying the improvement of cell adhesion and longer continuous monitoring of neuronal differentiation process by 2D-SPR observation. Figure 1

Non-Invasive and Continuous Monitoring of Differentiation Process into Cardiomyocytes By 2D-SPR Observation

Introduction Two-dimensional surface plasmon resonance (2D-SPR) measurement method has the advantages of noninvasive and real-time for observation of intracellular reactions. We have already applied this attractive method to monitor cell differentiation noninvasively.P19CL6, mouse embryonal carcinoma cells were cultured in a medium containing DMSO and 5-azacytidine to induce cardiac differentiation. 2D-SPR observation of the cells was then performed every two days to monitor intracellular refractive index change. The resonance angle of cell regions increased as the differentiation progressed. However, in previous study, cells in differentiation process were once detached from the well plate with trypsin treatment and replated on a gold chip and 2D-SPR measurement was done after one day. Therefore, we considered that the cells might be damaged by trypsin treatment exactly. Here, we aimed to realize noninvasive and continual 2D-SPR observation of the cells during cardiac differentiation process. In this experiment, the sensor chip chamber was covered with a gas permeable membrane and cardiac differentiation process from day 4 to day 14 was continually observed with a high resolution of SPR imager. Experimental method In this experiment, P19CL6 cells were cultured on a well plate during the period for formation of embryoid bodies from day 0 to day 4 in the presence of DMSO, and cardiac differentiation was induced on a gold chip from day 4 to day 14 in the presence of DMSO and 5-Azacitidine.The gold sensor chip chamber was covered with a gas permeable membrane to avoid contamination. 2D-SPR observations were carried out every two days to obtain the SPR images of cell regions and to monitor how the resonance angle changes at the cell regions during cardiac differentiation process. Result and discussion It was demonstrated that cardiac differentiation of P19CL6 cells on a gold chip was possible and the SPR images of cell regions were continually observed from day 4 to day 14 without contamination. Though the average of the resonance angles at cell regions decreased from day 4 to day 8, but it changed to increase from day 8 to day 12 and reached the maximum on day 12 and day 14. It is considered that intracellular proteins may be reformed to differentiate from embryonic cells into cardiomyocytes from the day 4 to day 8. The cells became cardiomyocyte-like on the day 10 and beating could be observed on a well plate from day 12.From these results we conclude that continual observation with a SPR imager is very promising method for noninvasive monitoring of cardiac differentiation. Figure 1

Thermodynamic and kinetic insights into the interaction of kynurenic acid with human serum albumin: Spectroscopic and calorimetric approaches

The interaction of neuroactive kynurenic acid (KYNA) with human serum albumin (HSA) was studied from both thermodynamic and structural point of view. This protein-drug interaction has high relevance in neuroscience, because the serum albumin-based KYNA-containing composites may act as a potential drug delivery system. Reversible binding of KYNA on HSA has been investigated under physiological conditions at various temperatures. The overall results of isothermal titration calorimetry (ITC), fluorescence spectroscopy and two-dimensional surface plasmon resonance (SPR) techniques showed similar trend for the temperature dependence of the estimated association constant (KA) values. Moreover, the enthalpy- (ΔH0), entropy- (ΔS0) and heat capacity changes (ΔCp) of KYNA-HSA complex formation were also calculated and interpreted according to the integrated van't Hoff equation. The results indicate low-affinity complex formation between the protein and the drug molecule, with two independent binding sites. The presence of the drug in the protein caused significant structural change with an α-helix content decrease from 69.7% to 51.2% proved by circular dichroism (CD) measurements. Theoretical calculations also support the experimentally suggested two independent binding sites theory stabilized by salt bridges and aromatic π-π stacking in the proposed binding positions.

Simulation study on comprehensive sensing enhancement of BlueP/MoS2- and BlueP/WS2-based fluoride fiber surface plasmon resonance sensors: analysis founded on damping, field, and optical power

Two-dimensional (2D) heterostructure-based fluoride fiber surface plasmon resonance (SPR) sensor designs are simulated and analyzed while emphasizing figure of merit (FOM) enhancement in the near-infrared (NIR) spectral region. Through simultaneous optimization of NIR wavelength and Ag layer thickness, exceptionally large FOM values of 15,650.75 RIU-1 and 12,409.30 RIU-1 are achieved for BlueP/MoS2-based and BlueP/WS2-based fiber SPR sensors, respectively. The results are explained in terms of tunable radiation damping, power loss, and corresponding field enhancement. These FOM values are significantly greater than recently reported sensors. The BlueP/MoS2-based sensor with 48.8nm Ag film and at 738.4nm wavelength provides an all-round large FOM.

The two-dimensional hybrid surface plasma micro-cavity

A hybrid surface plasma micro-cavity structure with a defect cavity is formed based on the two-dimensional surface plasmon resonance photonic crystal waveguide structure. A cell defect is introduced in the centre of the photonic crystal layer to build the hybrid surface plasma micro-cavity structure. This work is numerical based on the finite-difference time-domain method. The photon energy is confined to the micro-cavity and the photon energy is strongest at the interface between the insulating layer and the metal layer. The micro-cavity structure has a very small mode volume of sub-wavelength scale in the 1550 nm communication band. The value of Q/V is up to 7132.08 λ/n−3.

MicroRNA Biosensing with Two-Dimensional Surface Plasmon Resonance Imaging.

Two-dimensional surface plasmon resonance (2D-SPR) imaging, which provides a real-time, sensitive, and high-throughput analysis of surface events in a two dimensional manner, is a valuable tool for studying biomolecular interactions and biochemical reactions without using any tag labels. The sensing principle of 2D-SPR includes angular, wavelength, and phase interrogation. In this chapter, the 2D-SPR imaging technique is applied for sensing a target microRNA by its corresponding oligonucleotide probes, with sequence complementarity, immobilized on the gold SPR sensing surface. However, the low SPR signal due to intrinsic properties such as low molecular weight and quantity (pico-nanomolar) of the microRNA in clinical samples limits the direct detection of microRNA. Therefore, we developed a biosensing technique known as MARS (MicroRNA-RNase-SPR) assay, which utilizes RNase H to digest the microRNA probes enzymatically for fast signal amplification, i.e., in order to increase both the SPR signal and readout speed without the need for pre-amplification of target cDNA by polymerase chain reaction (PCR). Practically, we targeted microRNA hsa-miR-29a-3p, whose signature correlates to influenza infection, for rapid screening of influenza A (H1N1) patients from throat swab samples.

Two-Dimensional Surface Plasmon Resonance Imaging System for Cellular Analysis.

Optical biosensors based on surface plasmon resonance (SPR) phenomenon have received a great deal of attention in cellular analysis applications. Sensitive and high-resolution SPR imaging (SPRi) platforms are very useful for real-time monitoring and measurement of individual cell responses to various exogenous substances. In cellular analysis, mainstream SPR-based sensors have potential for investigations of cell responses under ambient conditions. Evaluations that account only for the average response of cell monolayers mask the understanding of precise cell-molecular interactions or intracellular reactions at the level of individual cells. SPR/SPRi technology has attracted a great deal of attention for detecting the response of cell monolayers to various substances cultivated on the gold sensor chip. To unleash the full strength of SPRi technology in complex cell bio-systems, the applied SPR imaging system needs to be sufficiently effective to allow evaluation of a compound's potency, specificity, selectivity, toxicity, and effectiveness at the level of the individual cell. In our studies, we explore the utility of high-resolution 2D-SPR imaging for real-time monitoring of intracellular translocation of protein kinase C (PKC), and detection of neuronal differentiation in live cells at the level of individual cells. The PC12 cell line, which is one of the most commonly used neuronal precursor cell lines for research on neuronal differentiation, was chosen as a nerve cell model. Two dimensional SPR (2D-SPR) signals/images are successfully generated. We have found that cells treated with the differentiation factor nerve growth factor (NGF) showed a remarkable enhancement of SPR response to stimulation by muscarine, a nonselective agonist of the muscarinic acetylcholine receptor.

Kinetic and Thermodynamic Evaluation of Kynurenic Acid Binding to GluR1270-300 Polypeptide by Surface Plasmon Resonance Experiments.

This work clearly demonstrates an evaluation process that is easily performed and is simply based on the fitting of temperature-dependent surface plasmon resonance (SPR) sensorgrams to provide detailed thermodynamic characterization of biologically relevant interactions. The reversible binding of kynurenic acid (KYNA) on human glutamate receptor (GluR1) polypeptide (GluR1270-300)-modified gold surface has been studied at various temperatures under physiological conditions by two-dimensional SPR experiments. The registered sensorgrams were fitted by using different kinetic models without application of any commercial software. Assuming that the association of GluR1270-300-KYNA complex is first order in both reactants, the association (ka) and dissociation (kd) constants as well as the equilibrium constants (KA) and the Gibbs free-energy change (ΔG°) were given at 10, 20, 30, and 40 °C. Moreover, the enthalpy (ΔH° = -27.91 kJ mol(-1)), entropy (ΔS° = -60.33 J mol(-1) K(-1)), and heat capacity changes (ΔCp = -1.28 kJ mol(-1) K(-1)) of the model receptor-ligand system were also calculated using a spreadsheet program. Negative values of ΔG° and ΔH° indicate the exothermic formation of a stable GluR1270-300-KYNA complex, because the |ΔH| > |TΔS| relation suggests an enthalpy-driven binding process. The negative ΔH° and ΔS° values strongly support the formation of a salt bridge between KYNA and the positively charged residues of the polypeptide (Arg, Lys) at pH 7.4, confirmed by molecular docking calculations as well.

Application of surface plasmon resonance imaging to monitoring G protein-coupled receptor signaling and its modulation in a heterologous expression system.

BackgroundG protein-coupled receptors (GPCRs) are ubiquitous surface proteins mediating various biological responses and thus, important targets for therapeutic drugs. GPCRs individually produce their own signaling as well as modulate the signaling of other GPCRs. Real-time observation of GPCR signaling and modulation in living cells is key to molecular study of biological responses and pharmaceutical development. However, fluorescence imaging, the technique widely used for this purpose, requires a fluorescent dye which may inhibit biological responses or a fluorescent-tagged target protein created through time-consuming genetic manipulation. In this study, we applied two-dimensional surface plasmon resonance (SPR) imaging to monitoring the translocation of protein kinase C (PKC), a major GPCR-coupled signaling molecule in the widely used HEK293 cell lines and examined whether the signaling of, and, modulation between heterologously expressed GPCRs can be measured without fluorescent labeling.ResultsWe cultured HEK293 cells on the gold-plated slide glass and evoked SPR at the interface between the cell’s plasma membrane and the gold surface with incident light. The translocation of activated native PKC to the plasma membrane is expected to alter the incident angle-SPR extent relation, and this could be detected as a change in the intensity of light reflection from the specimen illuminated at a fixed incident angle. Direct activation of PKC with 12-O-tetradecanoylphorbol-13-acetate increased the reflection intensity. This increase indeed reported PKC translocation because it was reduced by a pre-treatment with bisindolylmaleimide-1, a PKC inhibitor. We further applied this technique to a stable HEK293 cell line heterologously expressing the GPCRs type-1 metabotropic glutamate receptor (mGluR1) and adenosine A1 receptor (A1R). (RS)-3,5-dihydroxyphenylglycine, a mGluR1 agonist, increased the reflection intensity, and the PKC inhibitor reduced this increase. A pre-treatment with (R)-N6-phenylisopropyladenosine, an A1R-selective agonist suppressed mGluR1-mediated reflection increase. These results suggest that our technique can detect PKC translocation initiated by ligand binding to mGluR1 and its modulation by A1R.ConclusionsSPR imaging turned out to be utilizable for monitoring GPCR-mediated PKC translocation and its modulation by a different GPCR in a heterologous expression system. This technique provides a powerful yet easy-to-use tool for molecular study of biological responses and pharmaceutical development.

Targeting of the kynurenic acid across the blood–brain barrier by core-shell nanoparticles

Core-shell nanoparticles (CSNPs) were developed to get over therapeutic amount of kynurenic acid (KYNA) across the blood–brain barrier (BBB). Bovine serum albumin (BSA) was used as core for encapsulation of KYNA and the BSA/KYNA composite was finally encapsulated by poly(allylamine) hydrochloride (PAH) polymer as shell. In the interest of the optimization of the synthesis the BSA and KYNA interaction was studied by two-dimensional surface plasmon resonance (SPR) technique as well. The average size of d~100nm was proven by dynamic light scattering (DLS) and transmission electron microscopy (TEM), while the structure of the composites was characterized by fluorescence (FL) and circular dichroism (CD) spectroscopy. The in vitro release properties of KYNA were investigated by a vertical diffusion cell at 25.0°C and 37.5°C and the kinetic of the release were discussed. The penetration capacity of the NPs into the central nervous system (CNS) was tested by an in vitro BBB model. The results demonstrated that the encapsulated KYNA had significantly higher permeability compared to free KYNA molecules. In the neurobiological serial of in vivo experiments the effects of peripherally administered KYNA with CSNPs were studied in comparison with untreated KYNA. These results clearly proved that KYNA in the CSNPs, administrated peripherally is suitable to cross the BBB and to induce electrophysiological effects within the CNS. As the neuroprotective properties of KYNA nowadays are proven, the importance of the results is obvious.

マルチビームスプリッターを用いる二次元表面プラズモン共鳴センサーによる多成分同時イムノアッセイの検討

マルチビームスプリッターを用いる二次元表面プラズモン共鳴センサーによる多成分同時イムノアッセイの検討

Fabrication of a Dual-Enzyme Sensing System Based on Surface Plasmon Resonance Imager (SPRi) for Simultaneous Determination of Bio-Markers

Saccharides such as glucose, galactose and amino acids like lysine, phenylalanine are known as bio-markers of metabolic disorders, and several analytical methods have been developed to determine these bio-markers in blood and urine by enzymatic method, HPLC analysis and so on. Multi-biosensing systems for simultaneous determination of these bio-markers are presently expected to be necessary for troublesome diagnosis. We have previously proposed enzyme-sensors based on a two-dimensional surface plasmon resonance imager (2D-SPRi), which can sensitively monitor refractive index change of local region in the evanescent field on an Au chip. L-lysine sensor, L-glutamate sensor and galactose sensor has been developed each by combining oxidase reactions with 2D-SPR imaging of Os polymer-redox state. In this study, we aimed to develop a dual-enzyme sensor capable of the simultaneous detection of lysine and galactose by means of co-immobilizing LysOx/Os-HRP polymer spot and GalOx/Os-HRP polymer spot adjacently on the Au chip, which is divided into two sections.

Opposite angiogenic outcome of curcumin against ischemia and Lewis lung cancer models: in silico, in vitro and in vivo studies

The aim of this study was to investigate the angiogenic effects of curcumin on an ischemia and lung cancer model. To induce ischemia combined with lung cancer models, unilateral femoral arteries of C57BL/6 mice were disconnected on one side of the mouse and Lewis lung carcinoma (LLC) cells were xenografted on the opposite side. Angiogenic effects and underlying mechanisms associated with curcumin were investigated. Molecular target(s), signaling cascades and binding affinities were detected by Western blot, two-dimensional gel electrophoresis (2-DE), computer simulations and surface plasmon resonance (SPR) techniques. Curcumin promoted post-ischemic blood recirculation and suppressed lung cancer progression in inbred C57BL/6 mice via regulation of the HIF1α/mTOR/VEGF/VEGFR cascade oppositely. Inflammatory stimulation induced by neutrophil elastase (NE) promoted angiogenesis in lung cancer tissues, but these changes were reversed by curcumin through directly reducing NE secretion and stimulating α1-antitrypsin (α1-AT) and insulin receptor substrate-1 (IRS-1) production. Meanwhile, curcumin dose-dependently influenced endothelial cells (EC) tube formation and chicken embryo chorioallantoic membrane (CAM) neovascularization. Curcumin had opposite effects on blood vessel regeneration under physiological and pathological angiogenesis, which was effected through negative or positive regulation of the HIF1α/mTOR/VEGF/VEGFR cascade. Curcumin had the promise as a new treatment modality for both ischemic conditions and lung cancer simultaneously in the clinic.

<i>In vitro</i> Assembly Properties of Human Type I and II Hair Keratins

Multiple type I and II hair keratins are expressed in hair-forming cells but the role of each protein in hair fiber formation remains obscure. In this study, recombinant proteins of human type I hair keratins (K35, K36 and K38) and type II hair keratins (K81 and K85) were prepared using bacterial expression systems. The heterotypic subunit interactions between the type I and II hair keratins were characterized using two-dimensional gel electrophoresis and surface plasmon resonance (SPR). Gel electrophoresis showed that the heterotypic complex-forming urea concentrations differ depending on the combination of keratins. K35-K85 and K36-K81 formed relatively stable heterotypic complexes. SPR revealed that soluble K35 bound to immobilized K85 with a higher affinity than to immobilized K81. The in vitro intermediate filament (IF) assembly of the hair keratins was explored by negative-staining electron microscopy. While K35-K81, K36-K81 and K35-K36-K81 formed IFs, K35-K85 afforded tight bundles of short IFs and large paracrystalline assemblies, and K36-K85 formed IF tangles. K85 promotes lateral association rather than elongation of short IFs. The in vitro assembly properties of hair keratins depended on the combination of type I and II hair keratins. Our data suggest the functional significance of K35-K85 and K36-K81 with distinct assembly properties in the formation of macrofibrils.

Two-dimensional surface plasmon resonance imager: An approach to study neuronal differentiation

There is a growing demand for the development of a new bioanalytical technique that is capable of monitoring neuronal differentiation noninvasively, in real time, and without any fluorescent probes. In a previous article, we demonstrated that a high-resolution two-dimensional surface plasmon resonance (2D–SPR) imager was very useful to monitor cell response on chemical stimulation in which protein kinase C (PKC) translocation was related. In the current study, we focused on developing a new method for monitoring neuronal differentiation and examined the application of the high-resolution 2D–SPR imager to monitor neuronal differentiation noninvasively and by a label-free format. We successfully monitored the intracellular signal transduction, which was mainly translocation of PKC in PC12 cells by the 2D–SPR imager, and found that the cells treated with a differentiation factor, nerve growth factor (NGF), showed a remarkable enhancement of 2D–SPR response to muscarine, carbachol, and acetylcholine stimulation. The results demonstrated that 2D–SPR sensing is applicable to in situ assessment of neuronal differentiation and to studying the expression state of the specific receptors in the living state.

Real-time monitoring of intracellular signal transduction in PC12 cells by two-dimensional surface plasmon resonance imager

Real-time observation of intracellular process of signal transduction is very useful for biomedical and pharmaceutical applications as well as for basic research work of cell biology. The conventional methods used to observe intracellular reactions have not been convenient with several steps such as labeling and washing steps prior to the readout. Consequently, there is a critical need for label-free observation techniques for monitoring intracellular reactions. For feasible and reagentless observation of intracellular alterations in real time, we examined the use of a high-resolution two-dimensional surface plasmon resonance (2D–SPR) imager for monitoring of intracellular signal transduction that was mainly translocation of protein kinase C via local refractive index change in PC12 cells adhered on a gold sensor slide without any indicator reagent. PC12 cells were stimulated with KCl and phorbol-12-myristate-13-acetate (PMA, a protein kinase C [PKC] activator) at different concentrations in order to induce intracellular PKC translocation. 2D–SPR signal (reflection intensity change) is very consistent with the cellular response normally detected for these stimulants. Our results suggest that complex intracellular reactions could be real-time monitored and characterized by the 2D–SPR imager. It is further expected that signal transmission that was followed by the translocation of signaling proteins could be observed at the single cell level with the high-resolution 2D–SPR imager.

Living cell-based allergen sensing using a high resolution two-dimensional surface plasmon resonance imager

Recently, several papers indicated that the surface plasmon resonance (SPR) technique was available to monitor stimulation responses of mammalian cells adhered on sensor chips. On the other hand, the newly developed two-dimensional SPR (2D-SPR) imager system can obtain 2D-images of local refractive index change on the surface of a gold thin film. From these backgrounds, we expected that the 2D-SPR imager can visualize the individual response of many mammalian cells, simultaneously. Here, we report the observation of an allergenic response of a model mast cell, rat basophilic leukaemia cell (RBL-2H3), by using the high magnification 2D-SPR imaging system after pre-sensitization with 0.1 μg mL(-1) anti-dinitrophenyl immunoglobulin E (anti-DNP IgE). The response of the cells was successfully observed as the increment of the SPR signal (reflection intensity) upon stimulation with 0.1-1000 ng mL(-1) DNP-modified bovine serum albumin (DNP-BSA).