Measurement of purine release with microelectrode biosensors

Purinergic signalling departs from traditional paradigms of neurotransmission in the variety of release mechanisms and routes of production of extracellular ATP and adenosine. Direct real-time measurements of these purinergic agents have been of great value in understanding the functional roles of this signalling system in a number of diverse contexts. Here, we review the methods for measuring purine release, introduce the concept of microelectrode biosensors for ATP and adenosine and explain how these have been used to provide new mechanistic insight in respiratory chemoreception, synaptic physiology, eye development and purine salvage. We finish by considering the association of purine release with pathological conditions and examine the possibilities that biosensors for purines may one day be a standard part of the clinical diagnostic tool chest.     

A stereocomplex platform efficiently detecting antigen-antibody interactions

Ultrathin poly(methyl methacrylate) (PMMA) stereocomplex films with macromolecularly double-stranded regular nanostructures were prepared by layer-by-layer assembly of isotactic and syndiotactic PMMAs on solid surfaces. Antibodies were immobilized through the Fc region-capturing protein A, which had been physically adsorbed on the complex film, and the binding of antigens to immobilized antibodies was quantitatively investigated by the quartz crystal microbalance technique. Greater amounts of protein A with native forms were adsorbed on the complex film than those on conventional single-component PMMA films. Antibodies with high target-binding activities were also immobilized on the complex film. A greater amount of antigens could be detected on the complex film. The activity of protein A was maintained on the complex for a long time even within a dried state. The mechanism for the preservation of protein native forms on the complex surface was speculated by analyzing the physical adsorption of proteins with various secondary structures. Stereocomplex films can be utilized as novel coating nanomaterials for efficiently detecting protein-protein interactions.     

Investigation of osteoprotegerin interactions with ligands and antibodies using piezoelectric biosensors

Osteoprotegerin (OPG, osteoclastogenesis inhibitory factor) is a secretory glycoprotein involved as a soluble factor in the regulation of bone mass. OPG and its ligand (RANKL) levels in serum indicate the osteoclast formation activity. Alterations of the RANKL/OPG concentration ratio may be the cause of bone loss in many imbalances including osteoporosis, hypercalcaemia, metastatic osteolytic lesions and rheumatic bone degradation. The interactions of OPG with several antibodies were studied using the piezoelectric quartz crystal sensor. Monoclonal anti-OPG antibodies (5H3, 4E6H9 and OPG1.3) were immobilised on the sensing surface modified with covalently attached monolayer of protein A. Binding of both OPG standard and recombinant OPGFc chimeric protein was followed in real time. All antibodies were able to bind OPG and OPGFc, though in the case of MAb 4E6H9 the immunocomplexes dissociated quickly in the absence of OPG. Alternatively, biorecognition layers with RANKL were used. Two versions of the piezoelectric sensor for OPG were developed. The direct immunosensor was based on the antibody 5H3 and the affinity sensor employed the immobilised RANKL. The RANKL sensor exhibited poor reproducibility of results. For the immunosensor, the measuring range was 1.2-35 U/L of OPG. One analysis was completed within 15 min; the sensors were used repeatedly using regeneration with glycine buffer (pH 2.0). The developed immunosensor seems promising for rapid determination of osteoprotegerin in serum.     

BEID: Database for sequence-structure-function information on antigen-antibody interactions

The B-cell Epitope Interaction Database (BEID; http://datam.i2r.a-star.edu.sg/BEID) is an open-access database describing sequence-structure-function information on immunoglobulin (Ig)-antigen interactions. The current version of the database contains 164 antigens, 126 Ig and 189 Ig-antigen complexes extracted from the Protein Data Bank (PDB). Each entry is manually verified, classified, and analyzed for intermolecular interactions between antigens and the corresponding bound Ig molecules. Ig-antigen interaction information that is stored in BEID includes solvent accessibility, hydrogen bonds, non-hydrogen bonds, gap volume, gap index, interface area and contact residues. The database can be searched with a user-friendly search tool and schematic diagrams for Ig-antigen interactions are available for download in PDF format. The ultimate purpose of BEID is to enhance the understanding of the rules of engagement between antigen and the corresponding bound Ig molecules. It is also a precious data source for developing computational predictors for B-cell epitopes.     

Bilayer lipid membranes (BLM): Study of antigen-antibody interactions

Previous work of del Castillo and co-workers has shown that bilayer lipid membranes (BLM) can be used as transducers for detection of antigen-antibody reactions. The present experiments extend the previous work by incorporating complement into the BLM system. The results indicate that the antigen-antibody complex or the complement has no ability to affect the BLM system separately, but when carefully combined they will destabilize the BLM even at a much reduced concentration. Further development using the BLM as a tool for investigating immunological reactions is suggested.     

Application of surface plasmon resonance toward studies of low-molecular-weight antigen-antibody binding interactions

Methods for studying low-molecular-weight antigen-antibody binding interactions using surface plasmon resonance detection are presented. The experimental parameters most relevant to studies of low-molecular-weight antigen-antibody binding interactions are discussed. Direct kinetic analysis of the binding interactions is most informative, providing both apparent association and dissociation rate constants from which equilibrium constants can be calculated. Equilibrium analysis, including steady-state and solution affinity studies, offers an alternative approach to direct kinetic analysis when knowledge of the individual kinetic rate constants is not required or difficult to determine. The various methods are illustrated by studies of an anti-T(4) Fab fragment binding interaction with several thyroxine analogs. The methods utilized were dependent on the affinity of the interaction. The high-affinity anti-T(4) Fab fragment/l-T(4) binding interaction was evaluated using direct kinetic analysis. An intermediate affinity anti-T(4) Fab fragment/l-T(3) binding interaction was evaluated using a combination of direct kinetic analysis, steady-state analysis, and solution affinity analysis. The relatively weak anti-T(4) Fab fragment/l-T(2) binding interaction was evaluated using steady-state and solution affinity analysis protocols. Several thyroxine tracers that could not be immobilized to a biosensor surface were also evaluated via the solution affinity format. In cases where a given binding interaction was examined using multiple methods the results were comparable.     

Influence of biologically inspired nanometer surface roughness on antigen-antibody interactions for immunoassay-biosensor applications

Current research efforts to improve immunoassay-biosensor functionality have centered on detection through the optimal design of microfluidic chambers, electrical circuitry, optical sensing elements, and so on. To date, little attention has been paid to the immunoassay-biosensor membrane surface on which interactions between antibodies and antigens must occur. For this reason, the objective of the present study was to manipulate the nanometer surface roughness of a model immunoassay-biosensor membrane to determine its role on sensitivity and specificity. It was hypothesized that surface roughness characteristics similar to those used by the body's own immune system with B-lymphocyte cell membranes would promote antigen-antibody interactions and minimize non-specific binding. To test this hypothesis, polystyrene 96-well plate surfaces were modified to possess similar topographies as those of B-lymphocyte cell membranes. This was accomplished by immobilizing Protein A conjugated gold particles and Protein A conjugated polystyrene particles ranging in sizes from 40 to 860 nm to the bottom of polystyrene wells. Atomic force microscopy results provided evidence of well-dispersed immunoassay-biosensor surfaces for all particles tested with high degrees of biologically inspired nanometer roughness. Testing the functionality of these immunosurfaces using antigenic fluorescent microspheres showed that specific antigen capture increased with greater nanometer surface roughness while nonspecific antigen capture did not correlate with surface roughness. In this manner, results from this study suggest that large degrees of biologically inspired nanometer surface roughness not only increases the amount of immobilized antibodies onto the immunosurface membrane, but it also enhances the functionality of those antibodies for optimal antigen capture, criteria critical for improving immunoassay-biosensor sensitivity and specificity.     

Impedimetric evaluation for diagnosis of Chagas' disease: antigen-antibody interactions on metallic electrodes

A polypeptide chain formed by recombinant antigens, cytoplasmic repetitive antigen (CRA) and flagellar repetitive antigen (FRA) (CF-Chimera) of Trypanosoma cruzi, was adsorbed on gold and platinum electrodes and investigated by electrochemical impedance spectroscopy on phosphate buffer saline solutions (PBS) containing a redox couple. It was found that the adsorption is strongly sensitive to the oxide layer on the electrode surface. In the majority of the experiments the antigens retained their activity as observed through their interaction with sera from chronic chagasic patients. The results expressed in terms of the charge transfer resistance across the interface, indicate the viability of using the impedance methodology for the development of a biosensor for serological diagnosis of Chagas' disease.     

Detection of antigen-antibody interactions by surface plasmon resonance. Application to epitope mapping.

Surface plasmon resonance (SPR) detection requires no labeling of antigen or antibodies and allows quantification of two or more interacting molecular species. The automated SPR instrument used here consists of an optical detection unit, an integrated liquid handling unit, and an autosampler. A first molecule is immobilized to the dextran modified surface of the sensor chip. By sequential introduction, the stepwise formation of multimolecular complexes can then be monitored. A two-site binding assay which allows characterization of MoAb epitope specificities is described. A polyclonal rabbit anti-mouse IgG1 (RAMG1) immobilized to the dextran surface is used to capture the first MoAb from unprocessed hybridoma culture supernatants. After introducing the antigen, the ability of a second MoAb to bind to the antigen is tested. The analysis cycle which is fully automated can be performed more than 100 times using the same RAMG1 surface. Since the detection principle allows monitoring of each reactant in the consecutive formation of a multimolecular complex, multi-site binding experiments can be performed. Five MoAbs recognizing different epitopes on an antigen were shown to bind sequentially, forming a hexamolecular complex. MoAbs were further characterized by inhibition analysis using synthetic peptides derived from the primary structure of their antigen. As a model system MoAbs against recombinant HIV-1 core protein p24 were used in all experiments.     

Amplification of antigen-antibody interactions based on biotin labeled protein-streptavidin network complex using impedance spectroscopy.

Antibody was covalently immobilized by amine coupling method to gold surfaces modified with a self-assembled monolayer of thioctic acid. The electrochemical measurements of cyclic voltammetry and impedance spectroscopy showed that the hexacyanoferrate redox reactions on the gold surface were blocked due to the procedures of self-assembly of thioctic acid and antibody immobilization. The binding of a specific antigen to antibody recognition layer could be detected by measurements of the impedance change. A new amplification strategy was introduced for improving the sensitivity of impedance measurements using biotin labeled protein-streptavidin network complex. This amplification strategy is based on the construction of a molecular complex between streptavidin and biotin labeled protein. This complex can be formed in a cross-linking network of molecules so that the amplification of response signal will be realized due to the big molecular size of complex. The results show that this amplification strategy causes dramatic improvement of the detection sensitivity of hIgG and has good correlation for detection of hIgG in the range of 2-10 microg/ml.     

Synergistic substrates determination with biosensors

High sensitive biosensors for heterocyclic compounds determination were built using oxidases-catalyzed hexacyanoferrate(III) reduction in the presence of these compounds. As oxidases Aspergillus niger glucose oxidase and recombinant Microdochium nivale carbohydrate oxidase were used. The biosensors were build using graphite electrodes and entrapped solution of the oxidases. The sensitivity of the biosensors achieves 5.2-14.5 microA microM-1 cm-2. The detection limit of some heterocyclic compounds was 0.2 microM. The sensitivity of biosensors was 300-10,000 times larger in comparison to hexacyanoferrate(III). To background the scheme of biosensors action kinetics of synergistic substrates oxidation was investigated in homogenous solution. The measurements showed that the rate of the reduction of low reactive substrate (hexacyanoferrate(III)) increased due to synergistic action of high reactive substrates (oxidized heterocyclic compounds). The modeling revealed the limiting step of the process. The increase of hexacyanoferrate(III) reduction rate is determined by the rate of reduced enzymes interaction with oxidized heterocyclic compound. The oxidation of heterocyclic compounds (mediators) with hexacyanoferrate(III) does not limit the process. The analysis of macrokinetics of biosensors action showed that synergistic effect may be realized and high biosensors sensitivity may be achieved if diffusion module of the enzyme reaction with the oxidized mediator and of a cross reaction is larger than 0.5. The calculated relative sensitivity is about three times higher in comparison to experimentally determined that may be caused by the limited stability of oxidized heterocyclic compounds and/or some external diffusion limitation of substrates.     

Biosensor analysis of antigen-antibody interactions as a priority step in the generation of monoclonal bispecific antibodies

A biosensor system aimed at real-time measuring molecular interactions among label-free reactants has been used for a comparative analysis of the binding features (i.e., association-dissociation rates and affinity constants) as well as epitope mapping between bivalent monoclonal antibodies and the derived monovalent bispecific monoclonal antibody. The results show that observed different affinities between parental and derived bispecific antibodies concern the association rate constant, whereas the dissociation rate constants are unaltered. The apparent affinity-constant values determined by solid-phase radioimmunoassay yielded figures almost overlapping with those obtained with the biosensor instrument. The results of the present work indicate that the biosensor system has gained a key role not only as a tool for the study of antigen-antibody interactions, but also for setting up the reference parameters for the selection of the best candidates in the generation of bispecific monoclonal antibodies.     

Photoreversible antigen-antibody reactions

A monoclonal antibody (Z1H01) for an oligopeptide carrying an azobenzene group, was prepared under conditions where the azobenzene group is in the trans form. The antibody bound the hapten peptide effectively when the hapten peptide is in the trans form (K = 5 x 10(7) M-1), but the antibody released the hapten under irradiation with UV light where the hapten is in the cis form. The antibody bound the hapten again, when the hapten reverted to the trans form after irradiation with visible light.     

Calorimetric biosensors with integrated microfluidic channels

A microfluidic device capable of measuring real-time enthalpy changes of biochemical reactions and thermal properties of biological fluids is presented in this paper. The device consists of a freestanding microthermopile integrated with a glass microfluidic reaction chamber. The p-type polysilicon/gold microthermopiles fabricated on a 2 μm thick thermally isolated membrane showed a sensitivity of 0.94 V/W and a thermal time constant of less than 100 ms. Although the device is not restricted to enzymatic reactions, in this paper measurements of the heat of reaction from the catalytic action of glucose oxidase, catalase, and urease on glucose, hydrogen peroxide, and urea, respectively, are reported. Reactions were performed in open air using liquid batch testing and in enclosed fluidic reaction chamber by continuous flow experiments. A sensitivity of 53.5 μV/M for glucose, 26.5 μV/M for hydrogen peroxide and 17 μV/M for urea was obtained. Detection limit for glucose in the continuous flow mode is 2 mM (30 pmol). The aim of this work is to demonstrate the potential of the integrated calorimetric microfluidic device for fundamental thermodynamic studies in biochemical reactions. Using arrays of such devices with immobilized enzymes multi-analyte detection can be accomplished and the effects of interferents from competing substrates can be compensated. This paper presents the design, fabrication and initial testing results from such a microthermopile-based thermal biosensor.     

Protein-based voltammetric biosensors fabricated with nanomaterials

Protein-based voltammetric biosensors are sensors based on the electric communication between proteins and electrodes. Recently, more and more nanomaterials are utilized to assist the fabrication of such kind of biosensors. In this review, we mainly detail the biosensors constructed with different kinds of nanomaterials depending on their categories in the past two years.     

AFM force measurements of the gp120-sCD4 and gp120 or CD4 antigen-antibody interactions

Soluble CD4 (sCD4), anti-CD4 antibody, and anti-gp120 antibody have long been regarded as entry inhibitors in human immunodeficiency virus (HIV) therapy. However, the interactions between these HIV entry inhibitors and corresponding target molecules are still poorly understood. In this study, atomic force microscopy (AFM) was utilized to investigate the interaction forces among them. We found that the unbinding forces of sCD4–gp120 interaction, CD4 antigen–antibody interaction, and gp120 antigen–antibody interaction were 25.45 ± 20.46, 51.22 ± 34.64, and 89.87 ± 44.63 pN, respectively, which may provide important mechanical information for understanding the effects of viral entry inhibitors on HIV infection. Moreover, we found that the functionalization of an interaction pair on AFM tip or substrate significantly influenced the results, implying that we must perform AFM force measurement and analyze the data with more caution.