Pathogen detection using biosensors is commonly limited due to the need for sensitivity and specificity in detecting targets within mixed populations. These issues were addressed through development of a dual labeling method that allows for both liquid-phase fluorescence in situ hybridization (FISH) and capture antibody targeted detection (CAT-FISH). CAT-FISH was developed using Escherichia coli O157:H7 and Staphylococcus aureus as representative bacteria, and processing techniques were evaluated with regard to FISH intensities and antibody recognition. The alternative fixative solution, methacarn, proved to be superior to standard solid-phase paraformaldehyde fixation procedures, allowing both FISH labeling and antibody recognition. CAT-FISH treated cells were successfully labeled with FISH probes, captured by immunomagnetic separation using fluorescent cytometric array beads, and detected using a cytometric array biosensor. CAT-FISH treated cells were detectable with LODs comparable to the standard antibody-based technique, (~ 103 cells/ml in PBS), and the technique was also successfully applied to two complex matrices. Although immunomagnetic capture and detection using cytometric arrays were demonstrated, CAT-FISH is readily applicable to any antibody-based fluorescence detection platform, and further optimization for sensitivity is possible via inclusion of fluorescently tagged antibodies. Since the confidence level needed for positive identification of a detected target is often paramount, CAT-FISH was developed to allow two separate levels of specificity, namely nucleic acid and protein signatures. With proper selection of FISH probes and capture antibodies, CAT-FISH may be used to provide rapid detection of target pathogens from within complex matrices with high levels of confidence.

Contamination of fresh produce with Escherichia coli O157:H7 and other pathogens commonly causes food-borne illness and disease outbreaks. Thus, screening for pathogens is warranted, but improved testing procedures are needed to allow reproducible same-day detection of low initial contamination levels on perishable foods, and methods for detecting numerous pathogens in a single test are desired. Experimental procedures were developed to enable rapid screening of spinach for E. coli O157:H7 by using multiplex-capable immunological assays that are analyzed using biosensors. Detection was achieved using an automated electrochemiluminescent (ECL) assay system and a fluorescence-based cytometric bead array. Using the ECL system, less than 0.1 CFU of E. coli O157:H7 per gram of spinach was detected after 5 h of enrichment, corresponding to 6.5 h of total assay time. Using the cytometric bead array, less than 0.1 CFU/g was detected after 7 h of enrichment, with a total time to detection of less than 10 h. These results illustrate that both biosensor assays are useful for rapid detection of E. coli O157:H7 on produce in time frames that are comparable to or better than those of other testing formats. Both methods may be useful for multiplexed pathogen detection in the food industry and other testing situations.

Although nearly all newly derived water purification methods have improved the water quality in developing countries, few have been accepted and maintained for long-term use. Field studies indicate that the most beneficial methods use indigenous resources, as they are both accessible and accepted by communities they help. In an effort to implement a material that will meet community needs, two fractions of mucilage gum were extracted from the Opuntia ficus-indica cactus and tested as flocculation agents against sediment and bacterial contamination. As diatomic ions are known to affect both mucilage and promote cell aggregation, CaCl2 was studied in conjunction and compared with mucilage as a bacteria removal method. To evaluate performance, ion-rich waters that mimic natural water bodies were prepared. Column tests containing suspensions of the sediment kaolin exhibited particle flocculation and settling rates up to 13.2 cm/min with mucilage versus control settling rates of 0.5 cm/min. Bacillus cereus tests displayed flocculation and improved settling times with mucilage concentrations lower than 5 ppm and removal rates between 97 and 98% were observed for high bacteria concentration tests (>10(8) cells/ml). This natural material not only displays water purification abilities, but it is also affordable, renewable and readily available.

Food-borne and waterborne microbial pathogens are a potential problem in biowarfare and public health. Such pathogens can affect the health, combat readiness, and effectiveness of the warfighter in a battlefield environment and present potential threats to the civilian population through intentional or natural contamination of food and water. Conventional procedures to detect and identify microbial pathogens in food, water, and other materials can take days to perform and may provide inconclusive information. Research at the University of South Florida's Advanced Biosensors Laboratory (ABL) focuses on development of sample processing procedures and biosensor-based assays for rapid detection of biothreat agents. Rapid processing methods, including use of an automated concentrator of microorganisms in water, have been developed for complex matrix samples including ground beef, apple juice, produce, potable water and recreational water, enabling such samples to be directly tested by biosensor assays for target analytes. Bacillus atrophaeus spores and other bacteria can be concentrated from potable and recreational water at low levels with a dead-end hollow-fiber ultrafiltration concentration system. Target bacteria recovered by these processing procedures can be identified by evanescent wave, fiber optic biosensors or other detection platforms. Fiber optic biosensor assays have been improved to include subsequent PCR analysis and viability determination of captured target bacteria using broth enrichment and/or ATP luminescence.

In the last decade an extraordinary amount of research and development has focused on alleviating problems associated with contaminated water. With the majority of the World's population living on the brink of illness due to bacterial contamination in town water supplies, much of this attention has been focused on bacteria removal and sensors. Many current decontamination techniques are too technologically advanced for less developed countries, often resulting in their rejection by the societies they serve. Sensor work has also come across problems including poor sensitivity making it difficult to detect microorganisms at low concentrations. We have been testing a material extracted from the Opuntia ficus-indica cactus which could possibly address both of these problems in conjunction with one another. This material, referred to as cactus mucilage, has proven itself in the past as a viable flocculating agent for use in water contaminated with sediments and heavy metals. Flocculation tests, now focused on Bacillus cereus and Escherichia coli, have also given insight on the mucilage's ability to gather and concentrate bacterial contaminants from ion-rich water supplies. In columns with bacteria suspended in hard and soft water, flocculation begins immediately and is complete in approximately five to ten minutes with concentration rates of up to 99%. In addition to cleaning the water, the flocs formed with the mucilage could be removed from the water for sensor use. Cactus mucilage is an ideal material for water treatment and assessment because it is a naturally occurring, low cost material that is easy to obtain, process and use. Using this type of green chemistry, not only are bacteria concentrations significantly lowered in contaminated water, but also a highly concentrated volume of bacteria is produced that could potentially aid in biosensors.