Antibody-based Biosensors Research Articles

Impedance Biosensor Utilizing a Si Substrate Deposited by Wet Methods

We report an impedance biosensor utilizing a Si electrode created by wet chemical deposition atop 6061 Al alloy. The sensor electrode is created by galvanic/electroless Si deposition from an electrolyte containing 10 mM HF and 20 mM Na2SiF6 in 80 wt% formic acid, followed by antibody immobilization. The impedance response of the sensor electrode to increasing concentrations of peanut protein Ara h 1, a common food allergen, can be fit to an equivalent circuit containing three RC loops. The circuit element most sensitive to antigen binding is the charge transfer resistance, yielding a detection limit of 4 ng/mL. Biosensors that utilize electrochemical impedance spectroscopy have been employed with a wide variety of immobilized biomolecules, including antibodies, receptor proteins, aptamers, and ssDNA. These biomolecules must be immobilized atop a conductive and biocompatible substrate, which is most commonly accomplished by amide bond formation to carboxylate-terminated Au-thiol self-assembled monolayers. However, Au-thiol self-assembly chemistry has been reported to have inadequate stability for many applications, with a shelf life limited to days to weeks. In addition, most sensors need to be calibrated, which for antibodybased biosensors requires antibody unfolding. For this reason, durable chemistry for antibody immobilization is also needed for biosensor regeneration during such a calibration procedure. In addition to Au, other biocompatible substrate materials that have been employed for impedance biosensors include C, Si, Pt, Ti, and ITO. Si is intriguing as a biosensor substrate, since it is directly below C in the periodic table, so Si-C bonds are of comparable strength to C-C and Si-Si bonds. Additional advantages of Si substrates for biosensors include easier incorporation into ULSI devices and easier surface preparation relative to C. Room temperature combined galvanic and electroless deposition of compact Si films was recently reported from concentrated formic acid. Here these Si films are used for immobilization of the mouse monoclonal antibody to peanut protein Ara h 1, and subsequent impedance detection of the protein antigen. Peanuts are considered one of the most dangerous food allergens, with severe anaphylactic reactions causing over 100 fatalities in the United States alone each year. Nine possible allergens within peanuts have been identified, Ara h 1 to Ara h 8, and peanut oleosin, with Ara h 1 the most widely studied.

A Double-Sandwich ELISA for Identification of Monoclonal Antibodies Suitable for Sandwich Immunoassays.

The sandwich immunoassay (sELISA) is an invaluable technique for concentrating, detecting, and quantifying target antigens. The two critical components required are a capture antibody and a detection antibody, each binding a different epitope on the target antigen. The specific antibodies incorporated into the test define most of the performance parameters of any subsequent immunoassay regardless of the assay format: traditional ELISA, lateral-flow immunoassay, various bead-based assays, antibody-based biosensors, or the reporting label. Here we describe an approach for identifying monoclonal antibodies (mAbs) suitable for use as capture antibodies and detector antibodies in a sELISA targeting bacterial protein toxins. The approach was designed for early identification of monoclonal antibodies (mAbs), in the initial hybridoma screen.

Recombinant antibodies and their use in biosensors

Inexpensive, noninvasive immunoassays can be used to quickly detect disease in humans. Immunoassay sensitivity and specificity are decidedly dependent upon high-affinity, antigen-specific antibodies. Antibodies are produced biologically. As such, antibody quality and suitability for use in immunoassays cannot be readily determined or controlled by human intervention. However, the process through which high-quality antibodies can be obtained has been shortened and streamlined by use of genetic engineering and recombinant antibody techniques. Antibodies that traditionally take several months or more to produce when animals are used can now be developed in a few weeks as recombinant antibodies produced in bacteria, yeast, or other cell types. Typically most immunoassays use two or more antibodies or antibody fragments to detect antigens that are indicators of disease. However, a label-free biosensor, for example, a quartz-crystal microbalance (QCM) needs one antibody only. As such, the cost and time needed to design and develop an immunoassay can be substantially reduced if recombinant antibodies and biosensors are used rather than traditional antibody and assay (e.g. enzyme-linked immunosorbant assay, ELISA) methods. Unlike traditional antibodies, recombinant antibodies can be genetically engineered to self-assemble on biosensor surfaces, at high density, and correctly oriented to enhance antigen-binding activity and to increase assay sensitivity, specificity, and stability. Additionally, biosensor surface chemistry and physical and electronic properties can be modified to further increase immunoassay performance above and beyond that obtained by use of traditional methods. This review describes some of the techniques investigators have used to develop highly specific and sensitive, recombinant antibody-based biosensors for detection of antigens in simple or complex biological samples.

Antibody-based biosensors for environmental monitoring

Antibody-based biosensors provide sensitive and rapid analytical tools for the detection of a range of pathogens and associated toxins. In this paper, the recent progress in antibody-based biosensors for environmental monitoring is reviewed with particular emphasis on generation and immobilization methods of antibody capture probes. We also describe the current available data on antibody-based detection of pathogens and toxins. Optimal assay design and the strengths and limitations of current sensor technologies for detection of biological agents in environment on site are also discussed.

Probing the Functional Heterogeneity of Surface Binding Sites by Analysis of Experimental Binding Traces and the Effect of Mass Transport Limitation

Many techniques rely on the binding activity of surface-immobilized proteins, including antibody-based affinity biosensors for the detection of analytes, immunoassays, protein arrays, and surface plasmon resonance biosensors for the study of thermodynamic and kinetic aspects of protein interactions. To study the functional homogeneity of the surface sites and to characterize their binding properties, we have recently proposed a computational tool to determine the distribution of affinity and kinetic rate constants from surface binding progress curves. It is based on modeling the experimentally measured binding signal as a superposition of signals from binding to sites spanning a range of rate and equilibrium constants, with regularization providing the most parsimonious distribution consistent with the data. In the present work, we have expanded the scope of this approach to include a compartment-like transport step, which can describe competitive binding to different surface sites in a zone of depleted analyte close to the sensor surface. This approach addresses a major difficulty in the analysis of surface binding where both transport limitation as well as unknown surface site heterogeneity may be present. In addition to the kinetic binding parameters of the ensemble of surface sites, it can provide estimates for effective transport rate constants. Using antibody-antigen interactions as experimental model systems, we studied the effects of the immobilization matrix and of the analyte flow-rate on the effective transport rate constant. Both were experimentally observed to influence mass transport. The approximate description of mass transport by a compartment model becomes critical when applied to strongly transport-controlled data, and we examined the limitations of this model. In the presence of only moderate mass transport limitation the compartment model provides a good description, but this approximation breaks down for strongly transport-limited surface binding. In the latter regime, we report experimental evidence for the formation of gradients within the sensing volume of the evanescent field biosensor used.

Application of Electrochemical Biosensors for Detection of Food Pathogenic Bacteria

Current practices for preventing microbial diseases rely upon careful control of various kinds of pathogenic bacteria in food safety and environmental monitoring. The main disadvantages of conventional bacterial detection methods are the multistep procedure and long time requirements. This article gives an overview of alternative electrochemical biosensors for detection of pathogenic bacteria in the food industry. Focus has been on new microbial metabolism-based, antibody-based and DNA-based biosensors. The underlying principles and applications of these biosensors are discussed. Recent developments in flow-injection biosensor systems with an electrochemical detection are also presented.