Surface Plasmon Resonance Imaging Technique Research Articles

Surface Plasmon Resonance Imaging Studies for Proteolytic Hydrolysis of Proteins

Abstract Hydrolysis of various proteins was simultaneously studied using a surface plasmon resonance imaging technique. Poly-l-lysine has been found to be completely resistant to hydrolysis, while bovine serum albumin, casein, and lactate dehydrogenase are hydrolyzed at different rates under the attack of pepsin.

Surface plasmon resonance imaging (SPRI) system and real-time monitoring of DNA biochip for human genetic mutation diagnosis of DNA amplified samples

Nucleic acid-based biochips represent a promising tool for gene sequence analysis, especially for mutation detection. Surface plasmon resonance imaging (SPRI) technique allows kinetic monitoring of molecular interaction, such as DNA–DNA, in real-time and without any prior labelling step. SPRI was applied to developing a DNA sensor for the detection of gene mutations accounting for human cystic fibrosis and specifically some of those localized in exon 10, with the common three-base-pair deletion ΔF508, alongside several single nucleotide polymorphisms (SNPs). The SPRI system enabled us to monitor the hybridisation kinetics of unlabelled DNA targets (short oligonucleotides or a 377 pb PCR product) to a 196 spots matrix of ssDNA probes immobilised onto a bio-functionalised surface, and to detect in real-time the mutations in a DNA fragment.

Biosensor Device with SPR Imaging Technique: The Design of High Density DNA Bio-tip

We have developed a new methodology to fabricate high resolution DNA microarray (2,500-104 dots/cm2) in combination with micro contact printing (μ-CP) and SPR imaging. Novel COOH-terminated PEG-disulfides with p-carborane were synthesized to fabricate the first layer on gold substrates to achieve surface coupling reaction with amino-terminated DNA in high yield. The hybridization of the target-DNA modified gold nanoparticles on probe-DNA patterned surfaces was successfully demonstrated by the SPR imaging, where nonspecific adsorption was not observed to the array background. The gold nanoparticle arrays provide quite high contrast even at low surface coverage (∼10%) by the enhancement effect of optical signals based on nano-scale phenomena in the near field. This enhancement effect can be well demonstrated by the simulation based on Maxwell-Garnett (MG) theory and Fresnel's equation.

Label-Free Detection of Proteins in Crude Cell Lysate with Antibody Arrays by a Surface Plasmon Resonance Imaging Technique

We established a label-free method of measuring proteins in crude cell lysate using antibody arrays and surface plasmon resonance (SPR) imaging. The refractivity of the running buffer was adjusted with that of the lysate to overcome the bulk effect. The chemistries of the fabricated arrays were investigated to reduce nonspecific adsorption on the array surface. We found that the hydrophilicity of the poly(ethylene glycol) moiety and lower electrostatic charge on the surface provided a specific measurement of antigen-antibody interaction. We validated the system by measuring the expression of eight proteins in the mouse brain and comparing the results to those by conventional Western blotting. The detection limit of the antibody array was approximately 30 ng/mL in crude cell lysate, on the same order as that of previous SPR research. The system enabled quick, label-free, and high-throughput analysis of abundant proteins with minimal sample volume ( approximately 200 muL). It is expected that our SPR antibody array will be applicable for direct protein expression profiling of cell lysate, as well as for cell phenotyping, food analysis, discovery of new biomarkers, and immunological disease diagnostics.

Wavelength-scanning surface plasmon resonance imaging

A new surface plasmon resonance (SPR) imaging technique was proposed. After measurements were conducted at varying wavelengths, the wavelength affording the minimum brightness (SPR wavelength) was determined at each pixel of the image. A two-dimensional map of the SPR wavelength could be converted to a thickness profile by use of a nonlinear calibration curve, which was obtained by Fresnel calculation. An array of protein thin layers on a gold film was evaluated in air to present the layers' surface structure in nanometer scale.

Detection and Quantification of On-Chip Phosphorylated Peptides by Surface Plasmon Resonance Imaging Techniques Using a Phosphate Capture Molecule

We describe herein a detection and quantification system for on-chip phosphorylation of peptides by surface plasmon resonance (SPR) imaging techniques using a newly synthesized phosphate capture molecule (i.e., biotinylated zinc(II) complex). The biotinylated compound is a dinuclear zinc(II) complex that is suitable for accessing phosphate anions as a bridging ligand on the two zinc(II) ions. The compound was exposed on the peptide array and detected with streptavidin (SA) via a biotin-SA interaction by SPR imaging. In the conventional method using antibody, both anti-phosphoserine and anti-phosphotyrosine antibodies were required for phosphoserine and phosphotyrosine detection, respectively. Detection of the phosphate group by the zinc(II) complex, however, was independent of the phosphorylated amino acid residues. The calibration curve for the phosphorylation ratios was established with a calibration chip, on which phosphoserine-containing peptide probes were immobilized. The peptide probes, which were phosphorylated on the surface by protein kinase A, were detected and quantified by SPR imaging using the zinc(II) complex, SA, and anti-SA antibody. The reaction rate and the kinetics of on-chip phosphorylation were also evaluated with the peptide array. The phosphorylation ratio was saturated at approximately 20% in 2 h in this study.

Point mutation detection with the sandwich method employing hydrogel nanospheres by the surface plasmon resonance imaging technique

We propose a surface modification procedure to construct DNA arrays for use in surface plasmon resonance (SPR) imaging studies for the highly sensitive detection of a K- ras point mutation, enhanced with hydrogel nanospheres. A homobifunctional alkane dithiol was adsorbed on Au film to obtain the thiol surface, and ethyleneglycol diglycidylether (EGDE) was reacted to insert the ethyleneglycol moiety, which can suppress nonspecific adsorption during SPR analysis. Then streptavidin (SA) was immobilized on EGDE using tosyl chloride activation. Biotinylated DNA ligands were bound to the SA surface via biotin–SA interaction to fabricate DNA arrays. In SPR analysis, the DNA analyte was exposed on the DNA array and hybridized with the immobilized DNA probes. Subsequently, the hydrogel nanospheres conjugated with DNA probes were bound to the DNA analytes in a sandwich configuration. The DNA-carrying nanospheres led to SPR signal enhancement and enabled us to discriminate a K- ras point mutation in the SPR difference image. The application of DNA-carrying hydrogel nanospheres for SPR imaging assays was a promising technique for high throughput and precise detection of point mutations.

Analysis of μ-Contact Printed Protein Patterns by SPR Imaging with a LED Light Source

We demonstrate the characterization of mu-contact printed protein patterns and analysis of protein-protein interactions by two-dimensional (2-D) surface plasmon resonance imaging (SPRi). Advancements in SPRi image quality from employing a light emitting diode (LED) as the light source are described. We show that a LED offers an ideal point source that can eliminate interference artifacts and speckles found when using a laser source. The attainable thickness resolution in fixed-angle imaging is comparable to that of a monochromatic source, providing a solid foundation for quantitative analysis with the system. The SPR imaging technique reported here affords sub-nanometer thickness sensitivity and micrometer lateral resolution, allowing for convenient studies of biomolecular interactions and surface morphologies of ultrathin films. Spatially well-defined protein patterns of bacterial toxins were obtained by microcontact printing using a polydimethylsiloxane (PDMS) stamp on a functionalized self-assembled monolayer on Au. The influence of protein concentration in the inking solution on transfer efficiency was investigated, and a nonlinear correlation was observed between the solution concentration and the amount of protein immobilized on the surface. Quantitative analysis of protein interaction was performed with toxin-specific antibody, showing a concentration-dependent relationship that verifies the retention of biological activity of the protein after printing. The study demonstrates the feasibility and effectiveness of using LEDs as light sources in SPR imaging, opening doors for developing compact SPR instruments for direct, sensitive, and label-free detection of biohazardous molecules.

Surface plasmon resonance imaging analysis of hexahistidine-tagged protein on the gold thin film coated with a calix crown derivative

A surface plasmon resonance (SPR) imaging system was constructed and used to detect the hexahistidine-ubiquitin-tagged human parathyroid hormone fragment (His6-Ub-hPTHF(1–34)) expressed inEscherichia coli. The hexahistidine-specific antibody was immobilized on a thin gold film coated with ProLinkerTM B, a novel calixcrown derivative with a bifunctional coupling property that permits efficient immobilization of capture proteins on solid matrices. The soluble and insoluble fractions of anE. coli cell lysate were spotted onto the antibody-coated gold chip, which was then washed with buffer (pH 7.4) solution and dried. SPR imaging measurements were carried out to detect the expressed His6-Ub-hPTHF (1–34). There was no discernible protein image in the uninduced cell lysate, indicating that non-specific binding of contaminant proteins did not occur on the gold chip surface. It is expected that the approach used here to detect affinity-tagged recombinant proteins using an SPR imaging technique could be used as a powerful tool for the analyses of a number of proteins in a high-throughput mode.

Surface plasmon resonance imaging measurements of ultrathin organic films.

The surface-sensitive optical technique of surface plasmon resonance (SPR) imaging is used to characterize ultrathin organic and biopolymer films at metal interfaces in a spatially resolved manner. Because of its high surface sensitivity and its ability to measure in real time the interaction of unlabeled biological molecules with arrays of surface-bound species, SPR imaging has the potential to become a powerful tool in biomolecular investigations. Recently, SPR imaging has been successfully implemented in the characterization of supported lipid bilayer films, the monitoring of antibody-antigen interactions at surfaces, and the study of DNA hybridization adsorption. The following is included in this review: (a) an introduction to the principles of surface plasmon resonance, (b) the details of SPR imaging instrumental design, (c) a short discussion concerning resolution, sensitivity, and quantitation in SPR imaging, (d) the details of DNA array fabrication on chemically modified gold surfaces, and (e) two examples that demonstrate the application of the SPR imaging technique to the study of protein-DNA interactions.