Multianalyte detection of food pathogens/toxins through enzymatic bio-nanotransduction on an electrochemical biosensor array /by Josh R. Branen.

Enzymatic Bio-nanotransduction is a biological detection system based on the use of an enzyme (biological transducer) to change a specific biological recognition event into nucleic acid (RNA) nano-signal molecules. We used biological recognition elements (antibodies, DNA sequences) linked to DNA templates with T7 promoter regions for detection of specific target molecules. In vitro transcription of DNA templates bound to target molecules produced RNA nano-signals specific for every target in the sample. Discrimination of RNA nano-signals using a standard enzyme linked oligonucleotide fluorescence assay (ELOFA) provided a correlation between nano-signal profiles and target concentrations.;The system was capable of detecting and distinguishing three species of specific IgG molecules at a level of 0.2 ng. The system was also shown to be capable of detecting mixed protein and DNA targets. The flexibility of this system was substantiated through the simultaneous detection of Escherichia coli O157 microorganisms and Staphylococcal enterotoxin B (SEB) using a labeled secondary antibody approach. The system was capable of detecting and distinguishing three species of specific IgG molecules at a level of 0.2 ng. The system was also shown to be capable of detecting mixed protein and DNA targets. The flexibility of this system was substantiated through the simultaneous detection of Escherichia coli O157 microorganisms and Staphylococcal enterotoxin B (SEB) using a labeled secondary antibody approach.;By using primary antibodies to functionalize magnetic beads and to produce biological recognition elements (antibodies) conjugated to nano-signal producing DNA templates we were able to simultaneously detect Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium and Staphylococcal enterotoxin B. Using an optimized protocol we were able to estimate our limit of detection as 2.4 x 10{esc}p3{esc}s cfu/ml for E. coli O157:H7, 1.9 x 10{esc}p4{esc}s cfu/ml for Salmonella enterica serovar Typhimurium and 0.11 ng/ml for SEB with multi-analyte detection in buffer. We also examined the detection of targets in a model food product (milk) and provided evidence for the improvement of detection in milk using heat treatment.;We were able to extend these results to the development of a nucleic acid array electrochemical biosensor for multiplex detection of food pathogens/toxins. Estimated limits of detection were 1.2 x 10{esc}p4{esc}s cfu/ml for Escherichia coli O157:H7, 6 x 10{esc}p4{esc}s for Salmonella enterica serovar Typhimurium and 0.32 ng/ml for SEB. A threshold cut-off approach was established to show the effective detection of all three targets in mixed samples.Thesis (Ph. D., Microbiology, Molecular Biology and Biochemistry)--University of Idaho, September 2006

Magnetic Bead and Fluorescent Silica Nanoparticles Based Optical Immunodetection of Staphylococcal Enterotoxin B (SEB) in Bottled Water

Staphylococcal enterotoxins (SEs) are a major cause of food-borne diseases, most commonly SEs assayed immunologically with ELISA. An immunoassay based on fluorescein dye doped silica dioxide nanoparticles (F-SiNPs) and magnetic bead (MB) is described here for the detection of staphylococcal enterotoxin B (SEB). F-SiNPs have unique optical properties which make them attractive for biosensing. The water-in-oil (W/O) reverse microemulsion method was used for the synthesis of F-SiNPs (~ 95 nm of diameter). The F-SiNPs were characterized using SEM, TEM and FTIR spectroscopy. The detection of SEB is preformed in PBS buffer, and bottled drinking water using sandwich immunoassay format. Target analytes were captured using MBs modified with the antigen-specific “capture” antibody, and detected using F-SiNP labeled secondary antigen-specific antibody. We report a limit of detection down to 1 ng/mL SEB spiked sample in less than 2 hr assay time using fluorocount method. This study demonstrates the bio warfare agent SEB capture by magnetic beads and detection using F-SiNPs