Cytometry on a chip: cellular phenotypic and functional analysis using grating-coupled surface plasmon resonance

Grating-coupled surface plasmon resonance imaging (GCSPRI) is a method for the accurate assessment of both cell phenotype and function. In GCSPRI, cells and/or proteins of interest are flowed across antibodies immobilized on a gold-coated sensor chip. The surface of the chip is illuminated with monochromatic light that couples with surface plasmons in the gold. At a specific angle of incidence, the GCSPR angle, the maximum amount of coupling occurs. Shifts in the GCSPR angle can be correlated with refractive index changes following cell or analyte capture by the immobilized antibodies. In addition, GCSPRI can image the cells as they are being captured. GCSPRI's multiplexed format allows for the parallel assessment of up to 400 individual antibody regions. In this paper, we demonstrate GCSPRI's ability to identify cells and proteins of interest and compare results to a traditional flow cytometry system. This technology represents a fast and powerful method for the simultaneous assessment of cell phenotype and function.     

Intact-cell-based surface plasmon resonance measurements for ligand affinity evaluation of a membrane receptor

Toward future applications to the discovery of drugs against membrane receptors on pathological cells, an intact-cell-based surface plasmon resonance (SPR) methodology has been developed. The injection of a suspension of epidermal carcinoma A431 cells (5×10(7)cells/ml), as an analyte, generated clear SPR responses to epidermal growth factor (EGF) immobilized on the sensor chip. Because the responses were competitively reduced by the free ligand EGF, added to the analyte cell suspension, they certainly reflect the specific interaction of the immobilized EGF with the extracellular region of its receptor, which is highly expressed on the surface of the A431 cells.     

Compensation for loss of ligand activity in surface plasmon resonance experiments

The determination of equilibrium binding constants is an important aspect of the analysis of protein-protein interactions. In recent years surface plasmon resonance experiments (e.g., with a BIAcore instrument) have provided a valuable experimental approach to determining such constants. The standard method is based on measuring amounts of analyte bound at equilibrium for different analyte concentrations. During the course of a typical surface plasmon resonance experiment the measured equilibrium levels for a given analyte concentration often decrease. This appears to be due to a loss of activity of the protein coupled to the sensor chip or other phenomena. The loss in signal can lead to an erroneous determination of the equilibrium constant. A data analysis approach is introduced that aims to compensate for the loss of activity so that its influence on the results of the experiments is reduced.     

On-chip Escherichia coli culture, purification, and detection of expressed proteins

In a recent study, we reported the results of a rapid high-throughput expression analysis of the affinity-tagged proteins present in total cell lysates, using a surface plasmon resonance (SPR) imaging protein chip system. In this paper, we describe a novel method, which is able to sequentially carry out a recombinant Escherichia coli culture, as well as the detection and purification of the expressed proteins on a single microwell chip, fabricated on a two-dimensional thin gold film. Following the induction of the protein on the microwell chip, the E. coli cells were lysed on the chip via the addition of lysozymes, and the expressed glutathione S-transferase-fused green fluorescent protein (GST–GFP) was then purified on the chip via affinity interaction with the glutathionylated gold surface of the chip. Finally, the expressed protein was directly detected using the surface plasmon resonance (SPR) imaging system. This system saves a substantial amount of time, experimental resources, and labor, by allowing for the complicated and labor-intensive procedures inherent to the production of recombinant proteins to be conducted on a single microwell chip, simply and economically.M. Kim and S. Y. Lee contributed equally to this work.     

Angle-scanning SPR imaging for detection of biomolecular interactions on microarrays

In this paper we describe the use of a commercial surface plasmon resonance (SPR) imaging instrument for monitoring the binding of biomolecules on user-defined regions of interest of a microarray. By monitoring the angle shift of the SPR-dip using a continuous angle-scanning mode instead of monitoring the change in reflectivity at a fixed angle, a linear relationship with respect to the mass density change on the surface will remain over a wide dynamic angle range of 8 degrees. Peptides (2.4 kDa) and proteins (150 kDa) were both spotted on the same sensor chip to illustrate that both, low and high molecular weight ligands with initial large differences in off-set SPR angles, can be applied within the same experiment. By using a fluorescently labeled antibody, SPR results can be confirmed by means of fluorescence microscopy after completion of a SPR experiment. SPR imaging in angle-scanning operation provides sensitive, accurate, and label-free detection of analyte binding on microarrays containing different molecular weight ligands.     

A protein detection technique by using surface plasmon resonance (SPR) with rolling circle amplification (RCA) and nanogold-modified tags

Surface plasmon resonance (SPR) can detect molecules bound to a surface by subtle changes in the SPR angle. By immobilizing probes onto the surface and passing analyte solution through the surface, changes in SPR angle indicate the binding between analyte and probes. Detection of analyte from solution can be achieved easily. By using rolling circle amplification (RCA) and nanogold-modified tags, the signals of analyte binding are greatly amplified, and the sensitivity of this technique is significantly improved. Furthermore, this technique has potentials for ultra-sensitive detection and microarray analysis. In this paper, this detection technique is introduced and shown to have great amplification capability. Using 5 nm nanogold with 30 min of RCA development time, this proposed protein detection technique shows over 60 times amplification of the original signal.     

Detection of Bax protein conformational change using a surface plasmon resonance imaging-based antibody chip

We describe an antibody chip technology that uses a surface plasmon resonance (SPR) imaging system to examine the conformational change of a protein. In this study, we used Bax protein, a pro-apoptotic member of the Bcl-2 family of proteins, as a model protein to investigate the conformational alteration triggered by a TNF-related apoptosis-inducing ligand (TRAIL), a potent inducer of apoptosis. To develop the antibody chip for detecting the Bax conformational change, we immobilized Bax monoclonal antibody 6A7, which recognizes only a conformationally changed Bax protein on a gold surface. The resultant immobilized Bax antibodies provided specific and accurate measurements of the active conformation-specific epitope in the apoptotic cancer cells treated with the TRAIL; these measurements corresponded to the data obtained by immunoprecipitation analysis using an active conformation-specific Bax antibody (6A7). The results of our study indicated that TRAIL-induced Bax structural change could be monitored quickly and simply using an SPR imaging system, thus demonstrating the potential for using such a system for the analysis of conformational properties of target proteins.     

Design of buffer exchange surfaces and sensor chips for biosensor chip mass spectrometry

The feasibility of buffer exchange in biosensor chip mass spectrometry, along with the construction of base sensor chips and use of alternative chip chemistries, is demonstrated in this work. Beta-2-microglobulin (beta2m) was used as an analyte and captured in the first flow cell (FC1) on the sensor chip surface by an immobilized anti-beta2m antibody. Low pH buffer was then used to elute the captured analyte from the flow cell and route it to a second flow cell (FC2) downstream that served as a cation exchanger that retains the analyte. Following additional washes in FC1, the analyte present in FC2 was either eluted with a higher pH buffer (to demonstrate the possibility of elution into a downstream trypsin flow cell), or it was subjected to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry analysis to verify its presence in FC2. In a separate experiment, a gold-sputtered glass slide (base chip) was activated through a formation of 11-mercaptoundecanoic acid self-assembled monolayer and via reaction with 1,1"-carbonyldiimidazole. The activated chip was placed manually into the biosensor and two surfaces (flow cells) were derivatized with antibodies to beta2m and cystatin C (cysC). To evaluate the chip performance, diluted human urine aliquot was injected over the flow cells. Following the surface plasmon resonance analysis, the chip was MALDI-TOF MS analyzed, yielding signals from beta2m and cysC from their respective flow cells. Artifacts arising from the surface chemistries were not observed in the analysis.     

Whole cell-based surface plasmon resonance measurement to assess binding of anti-TNF agents to transmembrane target

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.     

In vitro farnesoid X receptor ligand sensor assay using surface plasmon resonance and based on ligand-induced coactivator association

Ligand binding to nuclear receptors leads to a conformational change that increases the affinity of the receptors to coactivator proteins. We have developed a ligand sensor assay for farnesoid X receptor (FXR) in which the receptor–coactivator interaction can be directly monitored using surface plasmon resonance biosensor technology. A 25-mer peptide from coactivator SRC1 containing the LXXLL nuclear receptor interaction motif was immobilized on the surface of a BIAcore sensor chip. Injection of the FXR ligand binding domain (FXRLBD) with or without the most potent natural ligand, chenodeoxycholic acid (CDCA), over the surface of the chip resulted in a ligand- and LXXLL motif-dependent interaction. Kinetic analysis revealed that CDCA and its conjugates decreased the equilibrium dissociation constant (K_d) by 8–11-fold, indicating an increased affinity. Using this technique, we found that a synthetic bile acid sulfonate, 3,7-dihydroxy-5β-cholane-24-sulfonate, which was inactive in a FXR response element-driven luciferase assay using CV-1 cells, caused the most potent interaction, comparable to the reaction produced by CDCA. This method provides a rapid and reliable in vitro ligand assay for FXR. This kinetic analysis-featured technique may be applicable to mechanistic studies.     

Kinetic analysis of adenovirus fiber binding to its receptor reveals an avidity mechanism for trimeric receptor-ligand interactions

Most adenoviruses bind to the N-terminal immunoglobulin domain D1 of the coxsackievirus and adenovirus receptor via the head part of their fiber proteins. Three receptor molecules can bind per fiber head. We expressed the D1 domain and the adenovirus type 2 fiber head in bacteria and studied binding interactions by surface plasmon resonance measurements. When receptor domains bind adenovirus fiber independently of each other, the dissociation constant is 20-25 nm. However, when adenovirus fiber binds to receptors immobilized on the sensor chip, a situation better mimicking adenovirus binding to receptors on the cell surface, the dissociation constant was around 1 nm. Kinetic analysis shows that this happens via an avidity mechanism; three identical interactions with high on and off rate constants lead to tight binding of one fiber head to three receptor molecules with a very low overall off rate. The avidity mechanism could be used by other viruses that have multimeric adhesion proteins to attach to target cells. It could also be more general to trimeric receptor-ligand interactions, including those involved in intracellular signaling.     

Surface plasmon resonance imaging-based protein array chip system for monitoring a hexahistidine-tagged protein during expression and purification

A surface plasmon resonance imaging-based Ni(2+)-iminodiacetic acid-coated gold chip system was developed to enable specific detection of a hexahistidine-tagged recombinant protein in crude extracts or in column chromatography fractions. This system is especially advantageous for high-throughput analysis of multiple proteins.     

Specific capture of mammalian cells by cell surface receptor binding to ligand immobilized on gold thin films

Aldehyde-terminated self-assembled monolayers (SAMs) on gold surfaces were modified with proteins and employed to capture intact living cells through specific ligand-cell surface receptor interactions. In our model system, the basic fibroblast growth factor (bFGF) binding receptor was targeted on baby hamster kidney (BHK-21) cells. Negative control and target proteins were immobilized on a gold surface by coupling protein primary amines to surface aldehyde groups. Cell-binding was monitored by phase contrast microscopy or surface plasmon resonance (SPR) imaging. The specificity of the receptor-ligand interaction was confirmed by the lack of cell binding to the negative control proteins, cytochrome c and insulin, and by the disruption of cell binding by treatment with heparitinase to destroy heparan sulfate which plays an essential role in the binding of bFGF to FGF receptors. This approach can simultaneously probe a large number of receptor-ligand interactions in cell populations and has potential for targeting and isolating cells from mixtures according to the receptors expressed on their surface.     

A disposable polymer sensor chip combined with micro-fluidics and surface plasmon read-out

This paper describes a new type of disposable polymeric sensor chip based on the grating coupling of surface plasmon modes combined with a micro-fluidic channel system. A specifically designed silicon stamp with nano-structure (grating) on the micro-structures (micro-channel) was fabricated by combining a holographic method and photolithography. By using such a stamp the micro-channels, the grating coupler and the gold which was first thermally evaporated onto the stamp were transferred to the polymeric substrate successfully in one step. It is demonstrated that the grating profile in the micro-channels allowed a very efficient coupling of the laser light to the surface plasmons propagating at the bottom of the micro-channels. The transferred gold exhibits properties of a freshly cleaned surface, and the self-assembly of a functional thiol derivative (mercapto-PEG) onto the sensor chip can be monitored by surface plasmon spectroscopy. The results obtained in this sensor chip show no difference from those obtained on a regular grating-coupled SPR sensor chip.     

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.     

Surface Plasmon Resonance Imaging-Based Protein Array Chip System for Monitoring a Hexahistidine-Tagged Protein during Expression and Purification

A surface plasmon resonance imaging-based Ni²⁺-iminodiacetic acid-coated gold chip system was developed to enable specific detection of a hexahistidine-tagged recombinant protein in crude extracts or in column chromatography fractions. This system is especially advantageous for high-throughput analysis of multiple proteins.     

Surface plasmon resonance imaging analysis of protein-protein interactions using on-chip-expressed capture protein

A surface plasmon resonance (SPR) imaging system, combined with a microwell gold chip for on-chip cell cultivation, was used to monitor protein-protein interactions. In particular, we developed an on-chip microscale cell cultivation system that integrates cell culture and on-chip analysis of protein-protein interactions on a single microwell chip in a time- and labor-saving manner. To assess the performance of this system in the analysis of protein-protein interactions, we conducted a series of protein-protein interaction analyses by measuring the binding of the yeast GAL4 dimerization domain (GAL4DD) to the GAL11 protein (GAL11P). Our system was found to enable the simple and rapid analysis of protein-protein interactions, requiring no special cell culturing equipment or recombinant protein expression prior to the immobilization of the purified proteins onto the chip. Our results demonstrate that the combination of an on-chip cell cultivation system and an SPR imaging system can be a useful tool to study protein-protein interactions without the need for time-consuming and labor-intensive protein preparation steps as well as fluorescent or other labeling of the interactants.     

An in situ electrochemical surface plasmon resonance immunosensor with polypyrrole propylic acid film: comparison between SPR and electrochemical responses from polymer formation to protein immunosensing

A novel in situ electrochemical surface plasmon resonance (EC-SPR) immunosensor is presented in this paper. The EC-SPR measurement can be used to in situ monitor the polymer formation, probe immobilization, antigen-antibody interaction and protein immunosensing process. A sandwich immunosensor based on permeable polypyrrole propylic acid (PPA) film is constructed using mouse IgG as a model analyte. The results show that the introduction of capture antibody conjugated enzyme not only enhances the current responses but also increases the SPR angle shift. The calibration curves of electrochemical (EC) and surface plasmon resonance (SPR) measurement exhibit a similar dependence on the bulk concentration of antigen. An approximate linear relationship can be obtained by plotting the data in semi-logarithmic reference frame. Compared with SPR, EC shows higher sensitivity with prolonged time. The in situ EC-SPR immunosensor described herein could have important potentials for diagnostics and medicine applications.     

Nanodiscs allow the use of integral membrane proteins as analytes in surface plasmon resonance studies

Nanodiscs are small-sized and flat model membranes that provide a close to native environment for reconstitution of integral membrane proteins. Incorporation of membrane proteins into nanodiscs results in water-soluble proteolipid particles making the membrane proteins amenable to a multitude of bioanalytical techniques originally developed for soluble proteins. The transmembrane domain of the human CD4 receptor was fused to ubiquitin with a preceding N-terminal decahistidine tag. The resulting integral membrane protein was incorporated into nanodiscs. Binding of the nanodisc-inserted histidine-tagged protein to a monoclonal anti-pentahistidine antibody was quantified using surface plasmon resonance (SPR) experiments. For the first time, a membrane-inserted transmembrane protein was employed as analyte while the antibody served as ligand immobilized on the sensor chip surface. SPR experiments were conducted in single-cycle mode. We demonstrate that the nanodisc-incorporated membrane protein showed nearly identical affinity toward the antibody as did the soluble decahistidine-tagged ubiquitin studied in a comparative experiment. Advantages of the new experimental setup and potential applications are discussed.