Jong Seol Yuk

Proteomic applications of surface plasmon resonance biosensors: analysis of protein arrays

Proteomics is one of the most important issues in the post-genomic area, because it can greatly contribute to identifying protein biomarkers for disease diagnosis and drug screening. Protein array is a key technology for proteome researches and has been analyzed by various methods including fluorescence, mass spectrometry, atomic force microscopy and surface plasmon resonance (SPR). SPR biosensor is a promising technology in proteomics, since it has various advantages including real-time measurement of biomolecular interactions without labeling and the simple optical system for the device. SPR biosensors have a strong potential for analyzing proteomes by SPR imaging and SPR spectroscopic imaging, even though the challenge is to produce proteins on a proteomic scale.

High-throughput analysis of mumps virus and the virus-specific monoclonal antibody on the arrays of a cationic polyelectrolyte with a spectral SPR biosensor

We investigated the potential use of a spectral surface plasmon resonance (SPR) biosensor in a high‐throughput analysis of mumps virus and a mumps virus‐specific mAb on the arrays of a cationic polyelectrolyte, poly(diallyldimethylammonium chloride) (PDDA). The PDDA surface was constructed by electrostatic adsorption of the polyelectrolyte onto a monolayer of 11‐mercaptoundecanoic acid (MUA). Poly‐L‐lysine was also adsorbed onto the MUA monolayer and compared with the PDDA surface in the capacity of mumps virus immobilization. The PDDA surface showed a higher adsorption of mumps virus than the poly‐L‐lysine surface. The SPR signal caused by the virus binding onto the PDDA surface was proportional to the concentration of mumps virus from 0.5 × 105 to 14 × 105 pfu/mL. The surface structure of the virus arrays was visualized by atomic force microscopy. Then, a dose‐dependent increase in the SPR signal was observed when various concentrations of the antimumps virus antibody in buffer or human serum were applied to the virus arrays, and their interaction was specific. Thus, it is likely that the spectral SPR biosensor based on the cationic polyelectrolyte surface may provide an efficient system for a high‐throughput analysis of intact virus and serodiagnosis of infectious diseases.

Sensitivity enhancement of spectral surface plasmon resonance biosensors for the analysis of protein arrays

A novel method for sensitivity enhancement of spectral surface plasmon resonance (SPR) biosensors was presented by reducing the refractive index of the sensing prism in the analysis of protein arrays. Sensitivity of spectral SPR biosensors with two different prisms (BK-7, fused silica) was analyzed by net shifts of resonance wavelength for specific interactions of GST–GTPase binding domain of p21-activated kinase-1 and anti-GST on a mixed thiol surface. Sensitivity was modulated by the refractive index of the sensing prism of the spectral SPR biosensors with the same incidence angle. The sensitivity of a spectral SPR biosensor with a fused silica prism was 1.6 times higher than that with a BK-7 prism at the same incidence angle of 46.2°. This result was interpreted by increment of the penetration depth correlated with evanescent field intensity at the metal/dielectric interface. Therefore, it is suggested that sensitivity enhancement is readily achieved by reducing the refractive index of the sensing prism of spectral SPR biosensors to be operated at long wavelength ranges for the analysis of protein arrays.

Array-Based Spectral SPR Biosensor: Analysis of Mumps Virus Infection

Spectral SPR biosensor is a useful system for a rapid analysis of protein arrays, as the biosensor with a fiber optic spectrometer can be easily aligned with the reflected light from protein arrays. The spectral SPR biosensor was constructed by Kretschmann geometry, based on the wavelength interrogation with various modules such as protein arrays, optical unit, programs for data acquisition and processing, and a motorized x-y stage for scanning. Protein arrays consist of glass/gold film/linker layer/protein/air. The surface of gold arrays was modified with poly(diallyldimethylammonium chloride) and 11-mercap-toundecanoic acid to immobilize mumps virus. The virus arrays were used to analyze anti-mumps virus in a buffer or human serum by the line-scanning mode of the spectral SPR biosensor. The array-based spectral SPR biosensor has a strong potential for a high-throughput serodiagnosis of infectious diseases.

Surface Plasmon Resonance Intensity in Ex Situ Analysis of Protein Arrays Using a Wavelength Interrogation-Based Surface Plasmon Resonance Sensor

We have investigated the properties of the surface plasmon resonance (SPR) spectra obtained by scanning each spot of protein arrays using a wavelength interrogation-based SPR sensor (custom-made). The surface structure of each protein array consists of glass/metal/proteins/air (ex situ). The analysis of serial spectra showed marked increases in SPR intensity and wavelength at the boundary area of protein array spots, which were mainly caused by the background signal from the glass substrate. However, at the central area, protein interaction caused an increase in SPR wavelength, and a decrease in SPR intensity. There was no significant difference between the central and boundary areas in terms of the roughness and thickness of the gold film. These results suggest that SPR intensity is a good indicator for distinguishing psuedosignals from real signals of biospecific interactions in the analysis of protein arrays using wavelength interrogation-based SPR sensors.

Development of an immunosensor with angular interrogation-based SPR spectroscopy

We present a self-constructed immunosensor with angular interrogation-based-surface plasmon resonance (SPR) spectroscopy in order to analyse antigen–antibody interactions. The SPR intensity, resonance angle and full width at half maximum (FWHM) were significantly affected by the thickness of an Au film. The optimal thickness for the Au film thickness as an active metal for surface plasmons was determined to be approximately 45 nm, as respects the SPR intensity and the FWHM of SPR spectrum. The detection limit of the sensor based on the minimal refractive index variation was found to be 1.4 × 10−4. The interaction of C-reactive protein (CRP) with anti-CRP on a 45 nm Au film was successfully analysed with the self-constructed SPR sensor. We suggest that the SPR sensor can be used as a useful tool for the analysis of immunoreactions.

Oriented Immobilization of C-Reactive Protein on Solid Surface for Biosensor Applications

An attempt of surface modification with receptor layers to achieve a maximal signal from antigen-antibody interaction on the solid surface has been undertaken. Interaction of C-reactive protein (CRP) with monoclonal anti-CRP has been investigated by comparative study by chemical cross-linking or electrostatic interaction in the framework of Layer-by-Layer approach. The processes of gold surface modification have been monitored by a wavelength interrogation-based surface plasmon resonance (SPR) sensor. Atomic force microscopy has been used for visualization of the surfaces modified with protein layers. The influence of biotinylated protein G-streptavidin (bPG/STV) complex on the SPR signal shift by antigen-antibody interaction has been studied. The influence of different cross-linking chemicals, such as di(N-succinimidyl)-3,3′-dithiodipropionate, 3-(2-pyridyldithio)-propionic acid N-hydroxysuccinimide ester, and N-hydroxysuccinimide/N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide on antigen immobilization of anti-CRP/bPG/STV system has been also examined. The film morphology of the first immobilized layer is very important for protein interactions. Maximum SPR-shift by CRP coupling with anti-CRP has been observed on the surface modified by streptavidin and di(N-succinimidyl)-3,3′-dithiodipropionate. AFM method can be used to directly monitor CRP/anti-CRP interaction on polyelectrolyte support.