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1.
Apoptosis can be routinely characterized using biomolecular markers such as in the TUNEL and the annexin V assays or by using fluorescent caspase substrates. Apoptosis can also be semi-quantitatively characterized using microscopy, which targets morphological features such as cell rounding, nuclear condensation and fragmentation as well as cell membrane blebbing. This label-free approach provides a limited resolution for the evolution of these events in time and relies heavily on subjective identification of the morphological features. Here we propose a label-free assay based on surface plasmon resonance (SPR) detection of minute morphology changes occurring as a result of apoptosis induction in an endothelial cell model (EA.hy926). At first, annexin V assays confirmed that our cellular model was responsive to TRAIL over a 12-hour period. Then, we show that SPR allows accurate monitoring of apoptosis by measuring (1) the duration of the latency period during which the apoptotic signal is integrated by the initiator caspases and transmitted to the executioner caspases, (2) the rate of the execution phase in which death substrates are cleaved and morphological changes occur, and (3) the total extent of apoptosis. Using these parameters, we characterized the responses obtained with TRAIL (EA.hy926, HeLa, AD-293) and the anti-Fas antibody (HeLa) for the extrinsic pathways and UV exposure (HeLa) for the intrinsic pathways. By comparing the SPR time-course of apoptosis with phase contrast micrographs, we demonstrate that the cell morphological hallmarks of apoptosis are the major contributors to the SPR signal. Altogether, our results validate the use of SPR as an accurate label-free assay for the real-time monitoring of apoptosis-triggered cell morphological changes.  相似文献   

2.
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.  相似文献   

3.
Optical sensors based on the excitation of surface plasmons, referred to as surface plasmon resonance (SPR) sensors, have become a central analytical tool for characterizing and quantifying a wide variety of macromolecular interactions, like receptor–ligand contacts. Besides this classical field of application, in the last 15 years, the development of SPR sensors aiming for the detection and analysis of ligand/cell or host/pathogen interactions, cell/cell contacts, and cellular reactions gained considerable momentum. The number of publications reporting about applications of SPR sensors implementing vital prokaryotic or eukaryotic cells as biorecognition elements for medical diagnostics, environmental monitoring, or biological safety is steadily growing. This review gives a short introduction to the technique of surface plasmon resonance and the parameters that are important for its application in the field of vital cell sensors. Furthermore, the publications concerning the application of such sensors in the analysis of cellular interactions and cellular reactions to extra- and intracellular stimuli are summarized.  相似文献   

4.
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.  相似文献   

5.
Line shape effects of the surface plasmon resonance (SPR) at the gold-water interface have been exploited, towards extending instrumental performance in the angular interrogation mode. Comprising a micro-fluidic set-up both, SPR-line broadening and associated signal symmetry/asymmetry features have been monitored in real time for selected adsorption systems and are compared with the simultaneously taken resonance angle that represents the commonly recorded SPR feature. Four angular broadening contributions have been identified: an intrinsic, temperature independent fraction, a temperature dependent part that dominates at ambient temperature, a surface morphology/roughness contribution and an adsorption related fraction. The latter, and substantially smaller contribution, is crucially affected by surface conditions, as the transition from hydrophobic to a hydrophilic surface state. Anionic sub-monolayer adsorption has been clearly resolved, along with detection of a pronounced calorimetric effect that originates from an exothermic surface reaction upon exposure of the protein covered gold surface to a strong chemical oxidant.  相似文献   

6.
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.  相似文献   

7.
We developed a technique for the measurement of surface plasmon resonance (SPR) to detect interactions of anti-tumor necrosis factor (TNF) agents with transmembrane TNF-α (mTNF-α) on living whole cells. The injection of a suspension of mTNF-α expressing Jurkat cells, used as an analyte, gave a clear binding response to anti-TNF agents, such as etanercept, infliximab and adalimumab, immobilized on sensorchip. The binding response of the analyte cells increased in a concentration-dependent manner and was competitively reduced by adding soluble TNF receptors to the analyte cell suspension. Treatment of analyte cells with free anti-TNF agent before injection reduced the binding response between the analyte cells and immobilized-etanercept on sensorchip, and the inhibitory effect of free anti-TNF agent was concordant with the affinity of anti-TNF agent for soluble TNF-α. These findings indicate that the SPR response arises from specific binding between anti-TNF agent and its target on cell membrane.  相似文献   

8.
Plasmonics - A surface plasmon resonance (SPR) biosensor based on a graphene nanoribbon array in a microfluidic flow cell operating in a flow-over format is studied. The optical response of the...  相似文献   

9.
Enzymatic activity monitoring of caspases, which are a class of cysteine protease, was performed by using peptide arrays based on surface plasmon resonance (SPR) imaging. The strategy of the detection is straightforward, using streptavidin to amplify the SPR signals of the surface-immobilized substrate peptides labeled with biotin at the C termini. Thus, the cleavage of the substrate peptides by caspases was detected as a signal decrease. Using this method, we succeeded in monitoring the activities of purified caspases and caspases in cell lysates. The SPR imaging-based peptide array would be applicable to cell-based drug screening and biochemical studies to reveal signal transduction processes.  相似文献   

10.
The present work reports an investigation of surface plasmon resonance (SPR) of silver nanoparticles in SiO2–TiO2 hosts. The surface plasmon resonance of silver nanoparticles was observed in the wavelength range 300–400 nm. Numerical calculation of SPR of silver nanoparticles with spherical morphology was done on the basis of discrete dipole approximation (DDA) method. The observed fluorescence spectrum fits well with the theoretically calculated one. The luminescence enhancement is attributed to the strong local electric field which increases the exciting and emitting photons coupled to SPR. An effort has been made to study the surface plasmon mediated excitation energy transfer (EET) between two spherical metal nanoparticles. The van der Waals (vdW) energy between plasmonic silver nanoparticles in the present hosts has been estimated.  相似文献   

11.
In this paper, we describe the detection of bacterial cell-extracted 16S ribosomal RNA (rRNA) using an emerging technology, surface plasmon resonance (SPR) imaging of DNA arrays. Surface plasmon resonance enables detection of molecular interactions on surfaces in response to changes in the index of refraction, therefore eliminating the need for a fluorescent or radioactive label. A variation of the more common SPR techniques, SPR imaging enables detection from multiple probes in a reusable array format. The arrays developed here contain DNA probes (15-21 bases) designed to be complementary to 16S rRNA gene sequences of Escherichia coli and Bacillus subtilis as well as to a highly conserved sequence found in rRNAs from most members of the domain Bacteria. We report species-specific hybridization of cell-extracted total RNA and in vitro transcribed 16S rRNA to oligonucleotide probes on SPR arrays. We tested multiple probe sequences for each species, and found that success or failure of hybridization was dependent upon probe position in the 16S rRNA molecule. It was also determined that one of the probes intended to bind 16S rRNA also bound an unknown protein. The amount of binding to these probes was quantified with SPR imaging. A detection limit of 2 micro g ml-1 was determined for fragmented E. coli total cellular RNA under the experimental conditions used. These results indicate the feasibility of using SPR imaging for 16S rRNA identification and encourage further development of this method for direct detection of other RNA molecules.  相似文献   

12.
The development of novel technologies capable of monitoring the dynamics of cell-cell and cell-substrate interactions in real time and a label-free manner is vital for gaining deeper insights into these most fundamental cellular processes. However, the label-free technologies available today provide only limited information on these processes. Here, we report a new (to our knowledge) infrared surface plasmon resonance (SPR)-based methodology that can resolve distinct phases of cell-cell and cell-substrate adhesion of polarized Madin Darby canine kidney epithelial cells. Due to the extended penetration depth of the infrared SP wave, the dynamics of cell adhesion can be detected with high accuracy and high temporal resolution. Analysis of the temporal variation of the SPR reflectivity spectrum revealed the existence of multiple phases in epithelial cell adhesion: initial contact of the cells with the substrate (cell deposition), cell spreading, formation of intercellular contacts, and subsequent generation of cell clusters. The final formation of a continuous cell monolayer could also be sensed. The SPR measurements were validated by optical microscopy imaging. However, in contrast to the SPR method, the optical analyses were laborious and less quantitative, and hence provided only limited information on the dynamics and phases of cell adhesion.  相似文献   

13.
We report on the application of surface plasmon resonance (SPR), based on Fourier transform infrared spectroscopy in the mid-infrared wavelength range, for real-time and label-free sensing of transferrin-induced endocytic processes in human melanoma cells. The evanescent field of the mid-infrared surface plasmon penetrates deep into the cell, allowing highly sensitive SPR measurements of dynamic processes occurring at significant cellular depths. We monitored in real-time, infrared reflectivity spectra in the SPR regime from living cells exposed to human transferrin (Tfn). We show that although fluorescence microscopy measures primarily Tfn accumulation in recycling endosomes located deep in the cell's cytoplasm, the SPR technique measures mainly Tfn-mediated formation of early endocytic organelles located in close proximity to the plasma membrane. Our SPR and fluorescence data are very well described by a kinetic model of Tfn endocytosis, suggested previously in similar cell systems. Hence, our SPR data provide further support to the rather controversial ability of Tfn to stimulate its own endocytosis. Our analysis also yields what we believe is novel information on the role of membrane cholesterol in modulating the kinetics of endocytic vesicle biogenesis and consumption.  相似文献   

14.
The labeling strategy with gold nanoparticles for the conventional surface plasmon resonance (SPR) signal enhancement has been frequently used for the sensitive determination of small molecules binding to its interaction partners. However, the influence of gold nanoparticles with different size and shape on SPR signal is not known. In this paper, three kinds of gold nanoparticles, namely nanorods, nanospheres, and nanooctahedrons with different size, were prepared and used to investigate their effects on the conventional SPR signal at a fixed excitation wavelength 670 nm. It was found that the SPR signal (i.e., resonant angle shift) was varied with the shapes and sizes of gold nanoparticles in suspension at a fixed concentration due to their different plasmon absorbance bands. For gold nanorods with different longitudinal absorbance bands, three conventional SPR signal regions could be clearly observed when the gold nanorod suspensions were separately introduced onto the SPR sensor chip surface. One region was the longitudinal absorbance bands coinciding with or close to the SPR excitation wavelength that suppressed the SPR angle shift. The second region was the longitudinal absorbance bands at 624 to 639 and 728 to 763 nm that produced a moderate increase on the SPR resonant angle shift. The third region was found for the longitudinal absorbance bands from 700 to 726 nm that resulted in a remarkable increase in the SPR angle shift responses. This phenomenon can be explained on the basis of calculation of the correlation of SPR angle shift response with the gold nanorod longitudinal absorbance bands. For nanospheres and nanooctahedrons, the SPR angle shift responses were found to be particle shape and size dependent in a simple way with a sustaining increase when the sizes of the nanoparticles were increased. Consequently, a guideline for choosing gold nanoparticles as tags is suggested for the SPR determination of small molecules with binding to the immobilized interaction partners.  相似文献   

15.
In vitro cell-based assays are widely used during the drug discovery and development process to test the biological activity of new drugs. Most of the commonly used cell-based assays, however, lack the ability to measure in real-time or under dynamic conditions (e.g. constant flow). In this study a multi-parameter surface plasmon resonance approach in combination with living cell sensing has been utilized for monitoring drug-cell interactions in real-time, under constant flow and without labels. The multi-parameter surface plasmon resonance approach, i.e. surface plasmon resonance angle versus intensity plots, provided fully specific signal patterns for various cell behaviors when stimulating cells with drugs that use para- and transcellular absorption routes. Simulated full surface plasmon resonance angular spectra of cell monolayers were compared with actual surface plasmon resonance measurements performed with MDCKII cell monolayers in order to better understand the origin of the surface plasmon resonance signal responses during drug stimulation of cells. The comparison of the simulated and measured surface plasmon resonance responses allowed to better understand and provide plausible explanations for the type of cellular changes, e.g. morphological or mass redistribution in cells, that were induced in the MDCKII cell monolayers during drug stimulation, and consequently to differentiate between the type and modes of drug actions. The multi-parameter surface plasmon resonance approach presented in this study lays the foundation for developing new types of cell-based tools for life science research, which should contribute to an improved mechanistic understanding of the type and contribution of different drug transport routes on drug absorption.  相似文献   

16.
Conformational changes of proteins immobilized on solid matrices were observed by measuring the adsorption of Triton X-100 (TX), a nonionic detergent, as a hydrophobic probe with BIACORE, a biosensor that utilizes the phenomenon of surface plasmon resonance (SPR). Two kinds of proteins, alpha-glucosidase and lysozyme, were covalently attached to dextran matrices on the sensor surface in the flow cell and then exposed to various concentrations of TX solution. We measured SPR signal changes derived from adsorption of TX to the immobilized proteins and calculated the monolayer adsorption capacity using the Brunauer-Emmett-Teller (BET) equation. The results demonstrated that monolayer adsorption capacity is proportional to the amount of immobilized proteins. Further, the unfolding process of immobilized proteins on the sensor surface induced by guanidine hydrochloride was investigated by monitoring SPR signal increases due to the adsorption of TX to the exposed hydrophobic region of the protein. Results strongly suggested that the increase in the SPR signal reflected the formation of the agglutinative unfolded state. We expect our measuring method using the SPR sensor and TX adsorption will be a novel tool to provide conformational information regarding various proteins on solid matrices.  相似文献   

17.
Immobilization is widely used to isolate agglutinative and associative proteins with large hydrophobic surfaces. Surface hydrophobicities of immobilized proteins were quantified by measuring the adsorption amounts of Triton X-100 as a hydrophobic probe with a biosensor that utilizes the phenomena of surface plasmon resonance (SPR). We measured SPR signal changes derived from adsorption of Triton X-100 to five kinds proteins and calculated the monolayer adsorption capacity using the Brunauer-Emmett-Teller equation, partly modified with a term for correcting an influence of the net charge of immobilized protein. SPR signal changes obtained by this method correlated with the values of surface hydrophobicities obtained by conventional assay using a hydrophobic probe. Thus this measuring method using an SPR sensor and Triton X-100 is expected to be a tool for quantifying surface hydrophobicities of immobilized proteins.  相似文献   

18.
Surface plasmon resonance (SPR)-based differential phase analysis has been presented. Real as well as complex plane analysis of resonance parameters have been undertaken for the optimum selection of metal thicknesses in a bimetallic SPR configuration working under both angular and spectral regime. Theoretically, we can characterize the aqueous solution in terms of this differential phase variation due to the variation of sample parameters such as concentration and temperature. In this respect, two case studies, namely, concentration of hemoglobin in human blood and sensing of temperature of water have been demonstrated and proposed theoretically. By monitoring the change of differential phase, proposed approach leads to a very sensitive measurement of concentration and temperature.  相似文献   

19.
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.  相似文献   

20.
Naphthyridine dimer is a unique molecule that strongly, and selectively, binds to the guanine-guanine mismatch in duplex DNA. We have synthesized naphthyridine dimers possessing a different length of poly(ethylene oxide) (PEO) linker, and immobilized them to CM5 sensor chip to carry out a surface plasmon resonance (SPR) assay of DNA duplexes containing a single base mismatch. The sensitivity of the sensor remarkably increased with increasing numbers of PEO units incorporated into the linker. With the sensor surface immobilized naphthyridine dimer for 1.5 x 10(3) response unit (RU) through three PEO units, the distinct SPR signal was observed at a concentration of 1 nM of the 27-mer G-G mismatch.  相似文献   

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