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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, (~10(3)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.

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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.

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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, CaCl(2) 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.

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One of the known limitations for biosensor assays is the high limit of detection for target cells within complex samples (e.g., Escherichia coli at 10(4) to 10(5) CFU/mL) due to poor capture efficiencies. Currently, researchers can only estimate the cell capture efficiency necessary to produce a positive signal for any type of biosensor using either cumbersome techniques or regression modeling. To solve this problem, green fluorescent protein (GFP) transformed E. coli O157:H7 was used to develop a novel method for directly and easily measuring the cell capture efficiency of any given biosensor platform. For demonstration purposes, E. coli-GFP was assayed on both fiber optic and planar waveguide biosensor platforms. Cells were enumerated using an epifluorescent microscope and digital camera to determine the number of cells captured on the surfaces. Conversion algorithms were used with these digital images to determine the cell density of entire waveguide surface areas. For E. coli-GFP, the range of cell capture efficiency was between 0.4 and 1.2%. This indicates that although the developed model works for calculating cell capture, there is still need for significant improvements in capture methods themselves, to increase the capture efficiency and thereby lower detection limits. The use of GFP-transformed target cells and cell capture efficiency calculations can facilitate the development and optimization processes by allowing direct enumeration of new biosensor design configurations and sample processing strategies.

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An intestinal protozoan parasite, Cryptosporidium parvum, is a major cause of waterborne gastrointestinal disease worldwide. Detection of Cryptosporidium oocysts in potable water is a high priority for the water treatment industry to reduce potential outbreaks among the consumer populace. Anti-Cryptosporidium oocyst polyclonal and monoclonal antibodies were tested as capture and detection reagents for use in a fiber optic biosensor assay for the detection of Cryptosporidium oocysts. Antibodies were validated using enzyme-linked immunosorbent assays, flow cytometry, Western blotting and fluorescent microscopy. Oocysts could be detected at a concentration of 105 oocysts/ml when the polyclonal antibodies were used as the capture and detection reagents. When oocysts were boiled prior to detection, a ten-fold increase in sensitivity was achieved using the polyclonal antibody. Western blotting and immunofluorescence revealed that both the monoclonal and polyclonal antibodies recognize a large (>300 kDa) molecular weight mucin-like antigen present on the surface of the oocyst wall. The polyclonal antibody also reacted with a small (105 kDa) molecular weight antigen that was present in boiled samples of oocysts. Preliminary steps to design an in-line biosensor assay system have shown that oocysts would have to be concentrated from water samples and heat treated to allow detection by a biosensor assay.