Real-time immunoassay of antibody activity in serum by surface plasmon resonance biosensor

A surface plasmon resonance biosensor has been used to determine antibody activity in serum. As a model system, the interaction of mouse IgG and sheep anti-mouse IgG polyclonal antibody was investigated in real time. The factors, including pH value, ionic strength, protein concentration, influencing electrostatic adsorption of mouse IgG protein onto carboxylated dextran-coated sensor chip surface, were studied. The procedures of mouse IgG protein immobilization and immune reaction were monitored in real time. The regeneration effect using the different elution reagents was also investigated. The same mouse IgG immobilized surface can be used for 100 cycles of binding and elution with only 0.38% loss per regeneration in reactivity. The results show that the surface plasmon resonance biosensor is a rapid, simple, sensitive, accurate and reliable detection technique for real-time immunoassay of antibody activity. The assay allows antibodies to be detected and studied in their native form without any purification. (C) 2000 Elsevier Science B.V. All rights reserved.

Detection of anti-human serum albumin antibody using surface plasmon resonance biosensor

The biosensor based on surface plasmon resonance(SPR) technology is a very useful tool to study the interaction between biomolecles. The main advantages of this technique is to "visualize" macromolecular interactions directly in real time, and in a label-free mode rather than indirect methods like enzyme-linked immunosorbent assays (ELISAs). We immobilize human serum albumin (HSA) to the carboxymethyldextran-modified sensor chip surface covalently to detect the activity of anti-HSA in serum, and regenerate the surface with .1 mol/L phosphoric acid. The results show that SPR biosensor can detect the activity of anti-HSA in real-time quickly and the sensor chip can be used over 100 cycles.

Studies of Gd-diethylenetriamine pentaacetic acid in boving serum albumin solution by nuclear magnetic resonance spectroscopy

In this paper, the water relaxation enhancement behavior of Gd-diethylenetriamine pentaacetic acid(DTPA) in water and in aqueous solution of bovine serum albumine(BSA) has been studied. The T-1 relaxivity of Gd-DTPA in BSA solution is higher than that in aqueous solution. The results indicate that Gd-DTPA can integrate non-covalently with BSA mainly in forms of (Gd-DTPA) . BSA, (Gd-DTPA)(2) . BSA, for which the apparent equilibrium constant is 0.026 mmol(-1).L,0.0018 mmol(-2).L-2 respectively. This method would be used to study the interactivities between protein and contrast agent.

Studies on electrostatic adsorption of proteins on modified surface by real-time surface plasmon resonance technique

The elucidation of key influence factors for electrostatic adsorption is very important to control protein nonspecific adsorption on modified surfaces. In this study, real-time surface plasmon resonance technique is used to characterize the electrostatic adsorption of two proteins (mouse IgG and protein A) on carboxymethyldextran-modified surface. The results show that protein solution pH and ionic strength are key influence factors for efficient electrostatic adsorption. The influence of protein, solution pH on the amount of electrostatic adsorption depends on the type of the charge and the charge density of both protein and modified matrix on the surface. The electrostatic adsorption process involves a competition between the positively charged protein and other positively charged species in the buffer solution. A decrease of ionic strength leads to an increasing electrostatic adsorption. The kinetic adsorption constants of protein A at different pH values were also calculated and compared.

Electrostatic layer-by-layer assembly of polycation and DNA multilayer films by real-time surface plasmon resonance technique

The assembly of alternating DNA and positively charged poly(dimethyldiallylammonium chloride) (PDDA) multilayer films by electrostatic layer-by-layer adsorption has been studied. Real time surface plasmon resonance (BIAcore) technique was used to characterize and monitor the formation of multilayer films in solution in real time continuously. The results indicate that the uniform multilayer can be obtained on the poly(ethylenimine) (PEI) coated substrate surface. The kinetics of the adsorption of DNA on PDDA surface was also studied by real-time BIAcore technique, and the observed rate constant was calculated using a Langmuir model (k(obs) = (1.28 +/- 0.08) x 10(-2) s(-1)).

Enhanced surface plasmon resonance immunosensing using a streptavidin-biotinylated protein complex

In this paper, we present a novel strategy for improving the sensitivity of surface plasmon resonance immunosensing using a streptavidin-biotinylated protein complex. This amplification strategy is based on the construction of a molecular complex between streptavidin and biotin labeled protein. The complex can be formed in a cross-linking network of molecules so that the amplification of the response signal will be realized due to the big molecular size of the complex. The results show that the amplification strategy causes a dramatic improvement of the detection sensitivity. hIgG protein could be detected in the range of 0.005-10 mug ml(-1).

Enzyme Colorimetric Assay Using Unmodified Silver Nanoparticles

Colorimetric assay based on the unique surface plasmon resonance properties of metallic nanoparticles has received considerable attention in bioassay due to its simplicity, high sensitivity, and low cost. Most of colorimetric methods previously reported employed gold nanoparticles (GNPs) as sensing elements. In this work, we develop a sensitive, selective, simple, and label-free colorimetric assay using unmodified silver nanoparticle (AgNP) probes to detect enzymatic reactions. Enzymatic reactions concerning adenosine triphosphate (ATP) dephosphorylation by calf intestine alkaline phosphatase (CLAP) and peptide phosphorylation by protein kinase A (PKA) were studied.

Fabrication and characterization of DNA/QPVP-Os redox-active multilayer film

Calf thymus DNA was immobilized on functionalized glassy carbon, gold and quartz substrates, respectively, by the layer-by-layer (LBL) assembly method with a polycation QPVP-Os, a quaternized poly(4-vinylpyridine) partially complexed with osmium bis(2,2'-bipyridine) as counterions. UV-visible absorption and surface plasmon resonance spectroscopy (SPR) showed that the resulting film was uniform with the average thickness 3.4 nm for one bilayer. Cyclic voltammetry (CV) showed that the total surface coverage of the polycations increases as each QPVP-Os/DNA bilayer added to the electrode surface, but the surface formal potential of Os-centered redox reaction shifts negatively, which is mainly attributed to the intercalation of redox-active complex to DNA chain. The electron transfer kinetics of electroactive QPVP-Os in the multilayer film was investigated by electrochemical impedance experiment for the first time. The permeability of Fe(CN)(6)(3-) in the solution into the multilayer film depends on the number of bilayers in the film. It is worth noting that when the multilayer film is up to 4 bilayers, the CV curves of the multilayer films display the typical characteristic of a microelectrode array.

Controlled nucleation and growth of surface-confined gold nanoparticles on a (3-aminopropyl)trimethoxysilane-modified class slide: A strategy for SPR substrates

The thickness of the gold film and its morphology, including the surface roughness, are very important for getting a good, reproducible response in the SPR technique. Here, we report a novel alternative approach for preparing SPR-active substrates that is completely solution-based. Our strategy is based on self-assembly of the gold colloid monolayer on a (3-aminopropyl)trimethoxysilane-modified glass slide, followed by electroless gold plating. Using this method, the thickness of films can be easily controlled at the nanometer scale by setting the plating time in the same conditions. Surface roughness and morphology of gold films can be modified by both tuning the size of gold nanoparticles and agitation during the plating. Surface evolution of the Au film was followed in real time by UV-vis spectroscopy and in situ SPRS. To assess the surface roughness and electrochemical stability of the Au films, atomic force microscopy and cyclic voltammetry were used. In addition, the stability of the gold adhesion is demonstrated by three methods. The as-prepared Au films on substrates are reproducible and stable, which allows them to be used as electrodes for electrochemical experiments and as platforms for studying SAMs.

Enhanced resonance light scattering based on biocatalytic growth of gold nanoparticles for biosensors design

The biocatalytic growth of gold nanoparticles (Au-NPs) has been employed in the design of new optical biosensors based on the enhanced resonance light scattering (RLS) signals. Both absorption spectroscopy and transmission electron microscopy (TEM) analysis revealed Au-NP seeds could be effectively enlarged upon the reaction with H2O2, an important metabolite that could be generated by many biocatalytic reactions.

Kinetic study of DNA/DNA hybridization with electrochemical impedance spectroscopy

The hybridization of immobilized oligonucleotides probe strands with solution phase targets is the underlying principle of microarraybased techniques for the analysis of DNA variation. To study the kinetics of DNA/DNA hybridization, target DNA is often prior labeled with markers. A label-free method of electrochemical impedance spectra (EIS) for study the hybridization in process was reported. The Langmuir model was used to determine the association rate constant (K-on), the dissociation rate constant (K-off) and the affinity rate constant (K-A), for perfect matched DNA hybridization. The results show that, EIS is a successful technique possessing high effectivity and sensitivity to study DNA/DNA hybridization kinetics. This work can provide another view on EIS for the studying of DNA/DNA hybridization.

Tunable optical properties of nano-Au on vanadium dioxide

The optical properties of Au nanoparticles deposited on thermochromic thin films of VO2 are investigated using spectroscopy. A localized modification on the transmittance spectrum of VO2 film is formed due to the presence of Au nanoparticles which exhibit localized surface plasmon resonance (LSPR) in the visible-near IR region. The position of the modification wavelength region shows a strong dependence on the Au mass thickness and shifts toward the red as it increases. On the other hand, it was found that the LSPR of Au nanoparticles can be thermally tunable because of the thermochromism of the supporting material of VO2. The LSPR wavelength, lambda(SPR), shifts to the blue with increasing temperature, and shifts back to the red as temperature decreases. A fine tuning is achieved when the temperature is increased in a stepwise manner.