James P. Whelan

Development and application of cell-based biosensors

AbstractBiosensors incorporate a biological sensing element that converts a change in an immediate environment to signals conducive for processing. Biosensors have been implemented for a number of applications ranging from environmental pollutant detection to defense monitoring. Biosensors have two intriguing characteristics: (1) they have a naturally evolved selectivity to biological or biologically active analytes; and (2) biosensors have the capacity to respond to analytes in a physiologically relevant manner. In this paper, molecular biosensors, based on antibodies, enzymes, ion channels, or nucleic acids, are briefly reviewed. Moreover, cell-based biosensors are reviewed and discussed. Cell-based biosensors have been implemented using microorganisms, particularly for environmental monitoring of pollutants. Biosensors incorporating mammalian cells have a distinct advantage of responding in a manner that can offer insight into the physiological effect of an analyte. Several approaches for transduction of cellular signals are discussed; these approaches include measures of cell metabolism, impedance, intracellular potentials, and extracellular potentials. Among these approaches, networks of excitable cells cultured on microelectrode arrays are uniquely poised to provide rapid, functional classification of an analyte and ultimately constitute a potentially effective cell-based biosensor technology. Three challenges that constitute barriers to increased cell-based biosensor applications are presented: analytical methods, reproducibility, and cell sources. Possible future solutions to these challenges are discussed.

Biological agent detection with the use of an airborne biosensor

The ability to identify aerosolized bacteria remotely with the use of a small unpiloted, all-electric aircraft was demonstrated. Swallow, an aircraft custom-built for the purpose of air-particle collection, was catapult-launched, flown by line of sight for 20-min missions, and recovered by landing on a short runway. Once airborne, the sensor payload, which included a particle collector, fluidics control unit, and biosensor, was activated. The sensor utilized was the Analyte 2000 fiber optic biosensor, which performs four simultaneous fluorescent sandwich immunoassays on the surface of tapered optical probes. Five-minute test cycles were conducted continuously and monitored at the ground station until the plane returned. Then Swallow and its sensor payload could be ready for additional flights within 30 min of landing. During the trial, Swallow successfully collected and identified an aerosolized bacterial sample.

KINETICS OF BINDING OF SMALL MOLECULAR WEIGHT ANTIGENS TO IMMOBILIZED ANTIBODIES IN FLOW

The continuous flow immunosensor has been described previously for detection of dinitrotoluene and cocaine [1,2]. Briefly, a Sepharose-immobilized antibody is saturated with fluorophore-labeled antigen and then placed into a continuous buffer flow stream. Introduction of unlabeled antigen into the flow stream results, within seconds, in displacement of labeled antigen, and subsequent detection downstream by fluorimetry. Detection levels for trinitrotoluene and cocaine have been measured in the low ng/ml range [2,3]. In addition to the exceptional sensitivity and rapid response time of the flow sensor, equally unexpected is that under these conditions, in the absence of added antigen, several days are required to significantly deplete the saturated column of labeled antigen at a flow rate of 1 ml/min. The focus of the present work is to address the apparent disparity between rapid displacement time and the abnormally long noncompetitive dissociation rates observed in the flow immunosensor. The standard flow immunosensor protocol employs an open-loop system which precludes measuring equilibrium constants directly for a given antibody/antigen pair. Such measurements can be closely approximated for immobilized antigens however, by flowing radiolabeled antigen in a closed loop over an antibody column until equilibrium is established. Once equillirium is obtained, ratios of bound-to-free antigen are used to calculate binding constants for a given antigen/antibody pair. Here, 3 H-cocaine is circulated over a column of Sepharose coupled with cocaine-specific monoclonal antibody until equilibrium is attained. Effects of flow rate on equilibrium constants will also be presented. Comparisons of association kinetics between immobilized antibodies in flow and antibodies in solution should provide a clearer understanding of the effects of both immobilization and flow on antibody binding and dissociation.