Determination of binding potency of peptidic inhibitors of Grb2-SH2 by using the protein-captured biosensor method

The growth factor receptor-binding protein 2-Src homology 2 (Grb2-SH2) domain plays an important role in the oncogenic Ras signal transduction pathway, therefore, peptidic inhibitors of the Grb2-SH2 domain has been chosen as our target for the development of antiproliferative agents. The inhibitory effects of peptide analogs on the Grb2-SH2 domain have been determined by using surface plasmon resonance (SPR) technology developed with the BIACORE biosensor. Recently, we reported the analysis of interactions between peptides and the GST-Grb2-SH2 that was immobilized on the surface of sensor chip by using BIACORE biosensor (the protein-immobilized method). Herein, we analyze interactions of peptides with the GST-Grb2-SH2 that was captured by the anti-GST antibodies immobilized on the surface of sensor chip (the protein-captured method). Results obtained by both methods are in good correlation, indicating the immobilization of GST-Grb2-SH2 on the sensor chip did not significantly affect the binding of Grb2-SH2 with peptides. Both SPR-based assays are very sensitive bioanalytical methods and can be applied in screening inhibitors of target proteins or purifying GST-fusion proteins, however, considering the efficiency and the cost, the GST-Grb2-SH2-immobilized method is suggested for routinely determining the binding potency of inhibitors of Grb2-SH2.     

BIACORE J: a new platform for routine biomolecular interaction analysis

SPR biosensor technology continues to evolve. The recently released platform from Biacore AB (Uppsala, Sweden), BIACORE J, is designed for the routine analysis of biomolecular interactions. Using an antibody-protein A and a ligand-receptor system, we demonstrate the utility of BIACORE J in determining active concentration and binding affinities. The results from these studies illustrate the high sensitivity of the instrument and its ability to generate reproducible binding responses. The BIACORE J is easy to operate and useful in diverse applications, making SPR technology widely accessible as a research tool.     

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.     

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.     

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.     

Probing the mechanism of drug/lipid membrane interactions using Biacore

Assay conditions were established to screen a panel of drugs for binding to liposome surfaces using a surface plasmon resonance (SPR) biosensor. Drugs were found to bind negligibly or reversibly or were retained on the liposome surface. Cationic amphiphilic drugs fell into the last class and correlated with drugs that induce phospholipidosis in vivo. To a first approximation, a single-site model yielded apparent binding affinities that adequately described a drug's dose-dependent binding to liposome surfaces. Affinities ranged at least 1000-fold within the drug panel. A liposome's drug-binding capacity and affinity depended on both the lipid headgroup and the drug's structure. Although a drug's charge state generally dominated whether or not it remained bound to the liposome, subtle structural differences between members of certain drug families led to them having widely differing binding affinities. A comparison between the dissociation of drugs from liposome surfaces by Biacore and the lipid retention measurements determined by a parallel artificial membrane permeability assay was drawn. The results from this study demonstrate the potential of using SPR-based assays to characterize drug/liposome-binding interactions.     

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.     

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.     

Immobilisation of DNA probes for the development of SPR-based sensing

An immobilisation procedure based on the direct coupling of thiol-derivatised oligonucleotide probes to bare gold sensor surfaces has been used for DNA sensing applications. The instrumentation used relies on surface plasmon resonance (SPR) transduction; in particular the commercially available instruments BIACORE X and SPREETA, have been employed in this study. The performances of the SPR-based DNA sensors resulting from direct coupling of thiol-derivatised DNA probes onto gold chips, have been studied in terms of the main analytical parameters, i.e. selectivity, sensitivity, reproducibility, analysis time, etc. A comparison between the thiol-derivatised immobilisation approach and a reference immobilisation method, based on the coupling of biotinylated oligonucleotide probes onto a streptavidin coated dextran sensor surface, using synthetic complementary oligonucleotides has been discussed. Finally, a denaturation method to obtain ssDNA ready for hybridisation analysis has been applied to polymerase chain reaction (PCR) amplified samples, for the detection of genetically modified organisms (GMOs).     

Using a Surface Plasmon Resonance Biosensor for Rapid Detection of Salmonella Typhimurium in Chicken Carcass

Chicken is one of the most popular meat products in the world. Salmonella Typhimurium is a common foodbome pathogens associated with the processing of poultry. An optical Surface Plasmon Resonance （SPR） biosensor was sensitive to the presence of Salmonella Typhimurium in chicken carcass. The Spreeta biosensor kits were used to detect Salmonella Typhimurium on chicken carcass successfully. A taste sensor like electronic tongue or biosensors was used to basically ＂taste＂ the object and differentiated one object from the other with different taste sensor signatures. The surface plasmon resonance biosensor has potential for use in rapid, real-time detection and identification of bacteria, and to study the interaction of organisms with dif- ferent antisera or other molecular species. The selectivity of the SPR biosensor was assayed using a series of antibody con- centrations and dilution series of the organism. The SPR biosensor showed promising to detect the existence of Salmonella Typhimurium at 1 x 106 CFU/ml. Initial results show that the SPR biosensor has the potential for its application in pathogenic bacteria monitoring. However, more tests need to be done to confirm the detection limitation.     

Rapid and label-free bacteria detection by surface plasmon resonance (SPR) biosensors

Surface Plasmon Resonance (SPR) biosensor technology has been successfully used for the detection of various analytes such as proteins, drugs, DNA, and microorganisms. SPR-based immunosensors that coupled with a specific antigen-antibody reaction, have become a promising tool for the quantification of bacteria as it offers sensitive, specific, rapid, and label-free detection. In this paper, we review the important issues in the development of SPR-based immunoassays for bacteria detection, concentrating on instrumentation, surface functionalization, liquid handling, and surface regeneration. In addition, this review touches on the recent advances in SPR biosensing for sensitivity enhancement.     

Evaluation of progesterone-ovalbumin conjugates with different length linkers in enzyme-linked immunosorbant assay and surface plasmon resonance-based immunoassay

A series of progesterone-4-ovalbumin (OVA) conjugates with different length linkers (4-, 11-, and 18-atoms long) were synthesized by successive aminocaproic acid homologation of 3-(pregn-4-ene-3,20-dione-4-yl)thiopropanoic acid (1) before conjugation to ovalbumin. The performance studies of these progesterone-4-ovalbumin conjugates showed that the effects of the length of linker on the antibody binding are dependent upon different immunoassay formats. In a rapid flow biosensor surface, on a BIAcore Surface Plasmon Resonance (SPR) instrument, antibody-binding capacities and response rate were dramatically increased for progesterone-4-ovalbumin conjugates when the length of the linker was incremented from 4 atoms to 11 or 18 atoms. Thus, highly sensitive SPR-based immunoassays for progesterone over a range of 0.1-50 ng ml(-1) were developed using biosensor surfaces immobilized with progesterone-ovalbumin conjugates having extended linkers. The SPR-based assays were fully competitive with conventional enzyme-linked immunosorbant assay (ELISA) but much more rapid and simple. However, there were little changes in antibody-binding performance using a conventional ELISA for the same conjugates. The progesterone-4-ovalbumin conjugate (1-OVA) had better antibody binding than its progesterone-7alpha-ovalbumin analog (2-OVA) in the SPR-based assay, but with a conventional ELISA there was no significant difference between these two isomeric conjugates.     

Signal enhancement of surface plasmon resonance immunoassay using enzyme precipitation-functionalized gold nanoparticles: a femto molar level measurement of anti-glutamic acid decarboxylase antibody

Colloidal gold nanoparticles (AuNPs) and precipitation of an insoluble product formed by HRP-biocatalyzed oxidation of 3,3'-diaminobenzidine (DAB) in the presence of H2O2 were used to enhance the signal obtained from the surface plasmon resonance (SPR) biosensor. The AuNPs were synthesized and functionalized with HS-OEG3-COOH by self assembling technique. Thereafter, the HS-OEG3-COOH functionalized nanoparticles were covalently conjugated with horseradish peroxidase (HRP) and anti IgG antibody to form an enzyme-immunogold complex. Characterizations were performed by several methods: UV-vis absorption, DLS, HR-TEM and FT-IR. The Au-anti IgG-HRP complex has been applied in enhancement of SPR immunoassay using a sensor chip constructed by 1:9 molar ratio of HS-OEG6-COOH and HS-OEG3-OH for detection of anti-GAD antibody. As a result, AuNPs showed their enhancement as being consistent with other previous studies while the enzyme precipitation using DAB substrate was applied for the first time and greatly amplified the SPR detection. The limit of detection was found as low as 0.03 ng/ml of anti-GAD antibody (or 200 fM) which is much higher than that of previous reports. This study indicates another way to enhance SPR measurement, and it is generally applicable to other SPR-based immunoassays.     

Real-time protein biosensor arrays based on surface plasmon resonance differential phase imaging

This paper reports the application of differential phase surface plasmon resonance (SPR) imaging in two-dimensional (2D) protein biosensor arrays. Our phase imaging approach offers a distinct advantage over the conventional angular SPR technique in terms of utilization efficiency of optical sensor elements in the imaging device. In the angular approach, each biosensor site in the biosensor array requires a linear array of optical detector elements to locate the SPR angular dip. The maximum biosensor density that a two-dimensional imaging device can offer is a one-dimensional SPR biosensor array. On the other hand, the phase-sensitive SPR approach captures data in the time domain instead of the spatial domain. It is possible that each pixel in the captured interferogram represents one sensor site, thus offering high-density two-dimensional biosensor arrays. In addition, our differential phase approach improves detection resolution through removing common-mode disturbances. Experimental results demonstrate a system resolution of 8.8 x 10(-7)RIU (refractive index unit). Real-time monitoring of bovine serum albumin (BSA)/anti-BSA binding interactions at various concentration levels was achieved using a biosensor array. The detection limit was 0.77 microg/ml. The reported two-dimensional SPR biosensor array offers a real-time and non-labeling detection tool for high-throughput protein array analysis. It may find promising applications in protein therapeutics, drug screening and clinical diagnostics.     

Initiation of Escherichia coli ribosomes on matrix coupled mRNAs studied by optical biosensor technique

The optical biosensor technique, based on the surface plasmon resonance (SPR) phenomenon, has been used to study the initiation of protein synthesis by E. coli ribosomes on surface coupled mRNA. mRNA was first periodate oxidized and then hydrazide coupled to the surface of a CM5 sensor chip. The formation of initiation complexes on the surface coupled mRNA was monitored in real-time with a BIACORE 2000 instrument. Mature 70S*mRNA*fMet-tRNA(Met) initiation complexes were assembled on mRNA by sequential introduction of the 30S and 50S subunits supplemented with appropriate initiation factors and fMet-tRNA(Met). We show that the formation of 70S*mRNA complexes on the surface coupled mRNA proceeds efficiently only in the presence of tRNA. Moreover, 70S*mRNA*fMet-tRNA(Met) complexes formed with fMet-tRNA(Met) are more stable than similar complexes formed with deacylated tRNAs. The efficient formation and slow dissociation of mature 70S*mRNA*fMet-tRNA(Met) initiation complexes are most easily explained by the stabilization of the interaction of the ribosomal subunits by fMet-tRNA(Met). This work demonstrates the feasibility of the BIACORE technique for studying the initiation of protein synthesis.     

Development and applications of surface plasmon resonance-based von Willebrand factor-collagen binding assay

Von Willebrand factor (vWf) functions both as a carrier of factor VIII (fVIII) in plasma and as an adhesive protein providing the primary link between collagen of the extracellular matrix and platelets sequestered from blood flow. The functional activity of vWf correlates with the level of its binding to collagen, which is commonly measured in the enzyme-linked immunosorbent assay (ELISA). We developed an automated collagen-binding assay employing the surface plasmon resonance (SPR) phenomenon, which allows one to quantitatively measure the binding of purified vWf and vWf-containing therapeutic fVIII concentrates to collagen type III immobilized on a biosensor chip. The results of the SPR-based assay highly correlated (r = 0.987) with collagen-binding ELISA. The advantages of the SPR-based assay are its higher accuracy and reproducibility in comparison with ELISA. We applied the developed assay for monitoring structural changes in the vWf component of plasma-derived fVIII/vWf concentrates during a virus inactivation procedure performed by heat treatment. We determined the critical residual moisture content of 2% that can be present in lyophilized concentrates during heat-treatment procedures without causing deteriorative changes in vWf properties. Our data suggest that the SPR-based assay is a useful tool in the development of industrial virus-inactivation procedures, allowing one to preserve vWf activity and achieve the maximal therapeutic efficacy of fVIII/vWf concentrates.     

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.     

Measuring adsorption of a hydrophobic probe with a surface plasmon resonance sensor to monitor conformational changes in immobilized proteins

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.     

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.     

Comprehensive Study of Phase-Sensitive SPR Sensor Based on Metal–ITO Hybrid Multilayer

Plasmonics - Surface plasmon resonance (SPR) biosensor is widely used for its high precision and real-time analysis. In this paper, SPR biosensor based on the hybrid structure of metal–indium...