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Array

The array biosensor is another optical biosensor that was originally developed at NRL (see RAPTOR and Integrating Waveguide Biosensor for others). After licensing to Hanson Technologies Inc. (Carlisle, PA), the technology was further developed and the version used in our lab is called the HLAB 5000. This biosensor operates similarly to the RAPTOR/Analyte 2000 in that a sandwich assay concept is employed for detection and laser light is used to excite a fluorescent label and generate a signal when targeted pathogens are present in the sample. However, the HLAB 5000 utilizes a matrix concept on a glass slide platform to permit detection of up to 12 different pathogens simultaneously. The HLAB 5000 has the capability to enable the development of automated continuous monitoring for air and water. Assays for the detection of Escherchia coli O157:H7 and Bacillus globigii spores have been developed using the HLAB 5000. A system for concentrating pathogens from drinking water using an automated filtration device and detecting them on the HLAB 5000 is under development in our laboratory.

Integrating Waveguide

The Integrating Waveguide Biosensor (IWB) is the most recent in a series of optical biosensors developed and patented by the Naval Research Laboratory (NRL). The IWB was preceded by the RAPTOR and Array Biosensor System (ABS). The IWB has been licensed to Creatv Microtech, Inc. (Potomac, MD) for further development into a commercial prototype. As with the other optical instruments, the IWB uses a sandwich immunoassay but the platform is the inside of a capillary tube. Unlike the RAPTOR or ABS, in which the exciting laser light traverses down the length of the waveguide fiber or slide (parallel to the waveguide), the laser of the IWB excites perpendicular to the waveguide (capillary) surface. The emitted fluorescence from the sandwich assay is coupled efficiently into the waveguide and is collected at the end of the tube where the signal is integrated by the sensing component. As a result of the physical configuration, the signal from a relatively large surface (internal portion of the capillary) is integrated and measured at a single point (end of capillary). The internal surface of the waveguide may be coated with target specific capture molecules such as antibodies, to create a “sensing surface” which can be used to capture and detect target analytes. Preliminary assays have been developed for Escherichia coli O157:H7 and Salmonella typhimurium.

RAPTOR

The RAPTOR and its laboratory-based version, the Analyte 2000, is a fiber optic biosensor that can rapidly detect pathogens directly from sample matrices with little or no sample preparation. It was originally developed by scientists at the Naval Research Laboratory (NRL) and was licensed to Research International (Monroe, WA) for further development and marketing. The biosensor detects organisms using a sandwich assay format. In this assay technique, antibodies or other pathogen-specific capture molecules (e.g., aptamer, peptide ligand or receptor) are layered on an optical fiber and used to first capture the target pathogen. The sample is run over the optical fibers and antibodies (or other capture molecules) labeled with a fluorescent dye (e.g., Cy5 or Alexafluor 633) are then used to detect the target. The biosensor uses a 635 nm laser light that excites the fluorophore and light emitted by the fluorophore is converted to a picoAmp signal that is recorded in the instrument. Increasing fluorescence signal is proportional to the target pathogen concentration. An assay can be run in a little as 15 minutes. The sensor is ideal for laboratory or field use as it is compact, portable, and can be set up for automatic operation in less than 15 minutes. A more recent version of the RAPTOR permits analysis of four different samples simultaneously. RAPTOR/Analyte 200 assays for Escherichia coli O157:H7 in ground beef, apple juice and drinking water and seawater, Salmonella typhimurium in sprout irrigation water, Bacillus. spores in powder and drinking water, Vibrio cholerae in oyster tissue, and Enterococci in seawater.

GloMax

The GloMax 96 is a microplate luminometer designed by Promega Corp. for a variety of cell-based bioluminescence assays adapted to 96-well plates. Bioluminescence assays measure light produced from chemical excitation, resulting in greater sensitivity than fluorescence assays by eliminating the fluorescent interference that can occur during photon-based excitation and emission. Bioluminescence assays are versatile and can be multiplexed, enabling the measurement of cellular changes associated with growth and viability, death, and cytotoxicity.  Cell viability is important to consider when assessing food, food product, and water contamination. Current research has focused on detecting and quantifying viable cells in complex samples such as ground beef, milk, and apple juice. Assays are being expanded to include produce wash as well as potable and recreational water. Future projects will explore the combination of viability assays with concentration methods as well as molecular detection methods.

ATP Bioluminescence Immunoassay to Detect Bacterial Pathogens
Tech ID#09A010