Gold Biosensor Research Articles

Monoclonal antibody--gold biosensor chips for detection of depurinating carcinogen--DNA adducts by fluorescence line-narrowing spectroscopy.

A new direct readout methodology for detection and quantitation of fluorescent carcinogen-DNA adducts is described. It combines the binding specificity of an immobilized monoclonal antibody (MAb) with high-resolution, low-temperature fluorescence spectroscopy. The MAb, which is covalently bound to a gold surface via a chemisorbed disulfide coupling agent, binds the adduct of interest in an aqueous sample. Laser-induced fluorescence under nonline narrowing (FNLN) and line-narrowing (FLN) conditions was used to detect (benzo[a]pyren-6-yl)guanine (BP-6-N7Gua) bound to immobilized MAb. At room temperature, the BP-6-N7Gua fluorescence was not detected, most likely because of quenching by the gold surface and/or efficient dynamical quenching. However, fluorescence was observed at room temperature when the surface was covered with a thin layer of glycerol, and possible reasons for the fluorescence enhancement are considered. Lowering of the temperature to 77 K led to nearly an order of magnitude increase in fluorescence intensity. Highly structured FLN spectra obtained at 4.2 K allowed for definitive adduct identification. The potential of this methodology for risk assessments of individuals exposed to polycyclic aromatic hydrocarbons is discussed.

Human GM-CSF interaction with the α-chain of its receptor studied using surface plasmon resonance

A surface plasmon resonance (SPR) based biosensor has been used for studying the interaction of recombinant human granulocyte–macrophage colony-stimulating factor (GM-CSF) with genetically engineered α-chain subunits of its specific receptor (GM-Rα). Western blot analysis of GM-Rα confirmed the correct size (80 kDa) and reactivity of these proteins against anti-GM-Rα polyclonal or monoclonal antibodies. GM-CSF was immobilized, using standard amine coupling methods, to the dextran-modified gold biosensor surface in order to capture GM-Rα subsequently injected over the sensing layer. GM-Rα were shown to specifically form complexes with the immobilized ligand. Pre-incubation of constant amounts of GM-Rα with dilutions of soluble GM-CSF before injection of the mixture over the GM-CSF matrix, prevented ligand binding in a dose dependent manner. In contrast, unrelated soluble cytokines or serum proteins (e.g. G-CSF, albumin, etc.) were found to exert no inhibition. Complexes formation blockage by pre-incubation of constant amounts of GM-Rα with dilutions of neutralizing anti-GM-Rα antibodies was concentration dependent, further assessing the specificity of the interaction. To investigate the possibility of relating the effect on binding affinity of critical conformational changes at the contact site, experiments of multisite binding were performed, flowing a set of neutralizing monoclonal antibodies reacting to different epitopes on GM-CSF over the GM-CSF matrix, before injecting GM-Rα. The results indicated that antibody interaction with helix D and helix A of GM-CSF markedly inhibited GM-CSF binding to GM-Rα. Comparable results were obtained using the biosensor technology and enzyme-linked immunoassays, in representative experiments performed with the same reagents. These experiments demonstrate that SPR can be successfully used for studying complementary interactions between GM-CSF and its receptor α-chains in solution without using labels or secondary tracers and, compared with conventional immunoanalysis methods, significantly saving time.

Sol−Gel-Derived Gold Composite Electrodes

The preparation, characterization, and analytical utility of sol-gel-derived gold composite electrodes is described. The new metal-ceramic electrodes are comprised of gold powder homogeneously dispersed in a modified silica matrix. They couple the favorable electron-transfer kinetics common to gold surfaces with the regeneration, bulk modification, and versatility features of sol-gel-derived composite materials. The voltammetric characteristics of the composite gold-silica electrodes are explored and compared with conventional gold electrodes. Sol-gel-derived gold biosensors have been prepared by incorporating an oxidase enzyme within the sol-gel gold solution. Analogous thick-film enzyme strips, based on a new screen-printable gold biogel ink, have also been fabricated. To our knowledge, the above represent the first examples of metal-ceramic sensing electrodes and of bulk modification of metallic working electrodes.

Measuring interactions of MHC class I molecules using surface plasmon resonance

To examine the molecular interactions between major histocompatibility complex (MHC)-encoded molecules and peptides, monoclonal antibodies (mAbs), or T cell receptors, we have developed model systems employing genetically engineered soluble MHC class I molecules (MHC-I), synthetic peptides, purified mAbs, and engineered solubilizable T cell receptors. Direct binding assays based on immobilization of one of the interacting components to the dextran modified gold biosensor surface of a surface plasmon resonance (SPR) detector have been developed for each of these systems. The peptide binding site of the MHC-I molecule can be sterically mapped by evaluation of a set of peptides immobilized through the thiol group of cysteine substitutions at each peptide position. Kinetic binding studies indicate that the MHC-I/peptide interaction is characterized by a low to moderate apparent k ass (∼ 5000–60000 M −1 s −1) and very small k dis (∼ 10 −4-10 −6 s −1) consistent with the biological requirement for a long cell surface residence time to permit engagement with T cell receptors. Several mAb directed against different MHC-I epitopes were examined, and kinetic parameters of their interaction with MHC molecules were determined. These showed characteristic moderate association rate constants and moderate dissociation rate constants ( k ass ∼ 10 4–10 6 M −1 s −1 and k dis ∼ 10 −2-10 −4 s −1), characteristic of many antibody/protein antigen interactions. The interaction of an anti-idiotypic anti-TCR mAb with its purified cognate TCR was of moderate affinity and revealed kinetic binding similar to that of the anti-MHC mAbs. The previously determined interaction of a purified T cell receptor with its MHC-I/peptide ligand is characterized by kinetic constants more similar to those of the antibody/antigen interaction than of the MHC-I/peptide interaction, but is remarkable for rapid dissociation rates (apparent k dis ∼ 10 −2 s −1). Such binding studies of reactions involving the MHC-I molecules offer insight into the mechanisms responsible for the initial specific events required for the stimulation of T cells.

MHC class I/peptide interactions: binding specificity and kinetics.

Recent developments in the preparation of soluble analogues of the major histocompatibility complex (MHC) class I molecules as well as in the application of real time biosensor technology have permitted the direct analysis of the binding of MHC class I molecules to antigenic peptides. Using synthetic peptide analogues with cysteine substitutions at appropriate positions, peptides can be immobilized on a dextran-modified gold biosensor surface with a specific spatial orientation. A full set of such substituted peptides (known as 'pepsicles', as they are peptides on a stick) representing antigenic or self peptides can be used in the functional mapping of the MHC class I peptide binding site. Scans of sets of peptide analogues reveal that some amino acid side chains of the peptide are critical to stable binding to the MHC molecule, while others are not. This is consistent with functional experiments using substituted peptides and three-dimensional molecular models of MHC/peptide complexes. Detailed analysis of the kinetic dissociation rates (kd) of the MHC molecules from the specifically coupled solid phase peptides reveals that the stability of the complex is a function of the particular peptide, its coupling position, and the MHC molecule. Measured kd values for antigenic peptide/class I interactions at 25 degrees C are in the range of ca 10(-4)-10(-6)/s. Biosensor methodology for the analysis of the binding of MHC class I molecules to solid-phase peptides using real time surface plasmon resonance offers a rational approach to the general analysis of protein/peptide interactions.