Immunomagnetic separation and rapid electrochemical detection strategies for microbial pathogens.

摘要

Biodefense, food safety, and water quality require the means to efficiently screen large volumes of samples for low concentrations of microbial pathogens. Rapid, sensitive, and field-ready detection methods are essential, but must also include a means to specifically extract and concentrate the target pathogen from a complex matrix. This thesis outlines three proof-of-concept approaches for rapid electrochemical detection of microbial pathogens, each beginning with immunomagnetic separation (IMS) of the target organism. Additionally, the development of an improved IMS methodology and its use in an electrochemical detection method are described.;In the first approach, target cells were detected directly by means of their ability to impede electrical current. Bacillus cereus (as a surrogate for B. anthracis) and E. coli O157:H7 were detected from pure culture with limits of 40 CFU/ml and 6 CFU/ml, respectively, in 65 min. In the second approach, Bovine Viral Diarrhea Virus (BVDV) from bovine serum samples was detected by means of an electroactive label, in 80 min. In the third approach, E. coli O157:H7 cells were electroactively labeled, magnetically positioned, and detected from pure culture with a limit of 70 CFU/ml (corresponding to 7 CFU present on the sensor) in 70 min.;Finally, an improved IMS methodology was developed for E. coli O157:H7 using two magnetic nanoparticle (MNP) types, and its specificity was initially evaluated against E. coli O55:H7 and Shigella boydii. The method, optimized in terms of antibody concentration, MNP concentration, and conjugation conditions, required only 35 min and yielded antibody-conjugated MNPs that were stable for up to 60 days.