Real time bio-warfare agent detection using bio-aerosol mass spectrometry

Chapter 1 introduces bio-warfare agents, their health risks and the necessity for rapid detection. Since the dissertation work focuses on real-time detection of Bacillus anthracis (anthrax), a Bacillus microbiology background is presented; including sporulation and germination. The effect of anthrax spore particle transport on disease spread is discussed. Mass spectrometry (MS) is presented, with an emphasis on bacteria identification. Equations for ion motion in a dual stage time-of-flight mass spectrometer (TOF-MS) are derived along with instrument design concepts. Matrix-assisted laser desorption/ionization (MALDI) is also introduced.; Chapter 2 presents real-time B. atropheus spore and vegetative cell detection by a first generation bio-aerosol mass spectrometer (BAMS). Biomarkers were identified by stable isotope labeling of whole organisms. Dipicolinic acid (DPA), amino acids, and phosphate adducts were identified. The low mass fingerprint spectra presented here, significantly limited real-time species level detection.; Chapter 3 investigates chemical factors governing ionization from micrometer particles, which helps explain why biomarkers identified in chapter 2 were observed. The rate at which standard amino acid, DPA, and mixture particles generate mass spectra was quantified by effective ionization probability (EIP). Aromatic constituents had the highest EIP, while non-aromatic had the lowest. Mixing aromatic DPA with non-aromatic arginine enhanced ionization of arginine. This supports the hypothesis that DPA acts as a natural MALDI matrix in spores, thereby aiding ionization of non-aromatic spore components.; In chapter 4, SIMION modeling was used to identify design flaws in the first generation BAMS, which yielded poor ion transmission at high m/z (>500Da). A novel BAMS was designed in SIMION and fabricated. A linear TOF with an ion guide was used to improve ion transmission. Delayed extraction was implemented to improve resolving power. The new design moved the BAMS mass range from ∼1,100Da to 36,000Da.; Chapter five presents the quantification of sensitivity for the new BAMS. Particles containing MALDI matrix and analyte were used. The new BAMS detected 14 zeptomoles (8,400 molecules) of gramicidin S; a new record in MALDI sensitivity. This high sensitivity will aid the detection of high mass biomarkers from single cells.