Robert A. Ogert

Detection of Clostridium botulinum toxin A using a fiber optic-based biosensor

A rapid, sensitive, analytical method for the detection of Clostridium botulinum toxin has been developed. The fiber optic-based biosensor utilizes the evanescent wave of a tapered optical fiber for signal discrimination. A 50 mW argon-ion laser, which generates laser light at 514 nm, is used in conjunction with an optical fiber probe that is tapered at the distal end. Antibodies specific for C. botulinum are covalently attached to the surface of the tapered fiber. The principle of the system is a sandwich immunoassay using rhodamine-labeled polyclonal anti-toxin A immunoglobin G (IgG) antibodies for generation of the specific fluorescent signal. Various anti-toxin antibodies were immobilized to the fibers. Affinity-purified polyclonal horse anti-toxin A antibodies performed better than the IgG fraction from the same horse serum or than the monoclonal anti-toxin A antibody BA11-3. Botulinum toxin could be detected within a minute, at concentrations as low as 5 ng/ml. The reaction was highly specific and no response was observed against tetanus toxin.

Detection of Cocaine Using the Flow Immunosensor

Abstract A continuous flow immunosensor has been designed for the detection of cocaine in aqueous samples. The continuous flow immunosensor relies on the displacement of fluorophore-labeled antigen from immobilized monoclonal antibody. The sensitivity and accuracy of the flow immunosensor were investigated while varying the parameters of immobilized antibody density, flow rate, amount of antibody-coated Sepharose used in each column, and the saturation of antibody binding sites with fluorophore-labeled antigen. Using a low density of immobilized anti-benzoylecgonine antibody, as little as 5 ng/ml cocaine could be detected. Small amounts of antibody-coated Sepharose could be used repeatedly and the lifetime of the column was proportional to the amount of Sepharose used. Results were obtained in less than a minute and cross-reactivity against various other drugs was negligible.

Kinetics of antibody binding at solid-liquid interfaces in flow

We have developed the theoretical framework for a displacement immunoassay conducted in flow under nonequilibrium conditions. Using a repetitive displacement technique, we determined the displacement rate and apparent dissociation rate constant at different flow rates. Our data suggest that the kinetics are best described by a first-order function. The displacement efficiency, the displacement rate, and therefore the apparent dissociation rate constant were calculated and demonstrated to be flow rate dependent. The theoretical framework developed in this study was successful in predicting the behavior of antigen displacement in flow.

A fiber-optic evanescent-wave immunosensor for large molecules☆

Abstract Detection of toxins and other large molecules is important for both clinical and environmental analyses. A significant factor in analyses of these compounds is the safety of the operator. The NRL fiber-optic biosensors has been created to utilize long optical fibers to prevent the contamination of the operator and the optical components from hazrdous materials. The optical fiber is clad for the majority of its length, with only a small portion of the core exposed. Capture antibodies are immobilized on the exposed core, which has been tapered for improved sensitivity. The toxins from Clostridium botulinum and pseudexin have been detected in the evanescent wave in concentrations as low as 30 pM in under 1 min.

Toxin detection using a fiber-optic-based biosensor

Using an evanescent wave fiber optic-based biosensor developed at Naval Research Laboratory, ricin toxin can be detected in the low ng/ml range. Sensitivity was established at 1 - 5 ng/ml using a two-step assay. The two-step assay showed enhanced signal levels in comparison to a one-step assay. A two-step assay utilizes a 10 minute incubation of an immobilized affinity purified anti-ricin antibody fiber optic probe in the ricin sample before placement in a solution of fluorophore-labeled goat anti-ricin antibodies. The specific fluorescent signal is obtained by the binding of the fluorophore-labeled antibodies to ricin which is bound by the immobilized antibodies on the fiber optic probe. The toxin can be detected directly from urine and river water using this fiber optic assay.

EVANESCENT WAVE FIBER OPTIC BIOSENSORS

The NRL fiber-optic biosensor was developed with the specific goal of using long fibers (1-20 m) to facilitate the analysis of environmental and clinical samples for hazardous materials. The long fibers were deemed important for preventing contamination of the operator and the optical components by potentially hazardous materials. The use of the biosensor is described here in terms of the chemistry for protein immobilization, stability of antibody-coated fibers, and sensitivity for detection of protein toxins. Antibodies coated on the fiber are stable for over 1 year of storage prior to use. The fiber optic biosensor has been used to measure ng/ml (pM) concentration of toxins in under a minute.

Comparative analysis of toxin detection in biological and enviromental samples

The basic recognition schemes underlying the principles of standard enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA) protocols are increasingly being adapted for use with new detection devices. A direct comparison was made using a fiber optic biosensor that employs evanescent wave detection and an ELISA using avidin-biotin. The assays were developed for the detection of Ricinus communis agglutinin II, also known as ricin or RCA60. Detection limits between the two methods were comparable for ricin in phosphate buffered saline (PBS), however results in complex samples differed slightly. In PBS, sensitivity for ricin was 1 ng/ml using the fiber optic device and 500 pg/ml using the ELISA. The fiber optic sensor could not detect ricin directly in urine or serum spiked with 5 ng/ml ricin, however, the ELISA showed detection but at reduced levels to the PBS control.

Evanescent-wave fiber optic biosensor: challenges for real-world sensing

We have developed an evanescent wave fiber optic biosensor which uses long fibers to facilitate the analysis of environmental and clinical samples for hazardous materials. The use of antibody/antigen binding with fluorescence-based sensing in the evanescent wave yields a sensor that is unique, adaptable, and sensitive. The variety of substances that could be detected is limited only by their antigenicity. Sensing in the real world poses several challenges that must be met. We have focused on the development of several aspects of the sensing system to transition this sensor into a field deployable device. Recent developments presented here include optimized fiber optic probe tapering, a flow chamber to facilitate sampling, and probe regeneration for repetitive analysis. Preliminary experiments assessing the potential to detect analytes in biological and environmental fluids are also presented.

Progress in Fiber-Optic Based Biosensors at the United States Naval Research Laboratory

A fiber-optic based biosensor has been developed which integrates a novel array of biological, optical, and electrical components. Distally tapered, chemically activated glass fibers are coated with antibodies specific for desired analytes. A sandwich immunoassay is performed by exposing a fiber to a solution of analyte containing a second, analyte-specific and fluorescently labeled antibody. Fluorescent light emitted from antibody/analyte complexes bound within the evanescent region of the laser illuminated tapered fiber is optically filtered and electronically quantitated. Assays for botulism and ricin toxins, as well as specific detection of fluorescently-stained Bacillus anthracis cells, are described.