Focused Microwaves for Cellular Lysing, DNA/RNA and Protein/Enzyme Fragmentation and Inactivation

Abstract

Rapid sample preparation is one of the main hindrances to infectious bacterial identification. To aid in rapid and efficient sample preparation, gold microwave lysing triangles (MLTs) was developed. Gold MLTs serve as a platform to focus microwaves directly to the center of a sample subsequently increasing the electromagnetic energy and temperature. This increase in energy and temperature allows for rapid cellular lysis followed by DNA/RNA fragmentation as well as protein/enzyme degradation and substantially lower activity. The first part of this research explores alterations to gold MLTs to ultimately lower the cost of the technology. Various alterations were investigated including but not limited to � the metal that was used, the microwave focusing geometry, and the gap size (distance) between the geometries. Additionally, standard microwave conditions were determined for Gram-positive and �negative bacteria, in particular L. monocytogenes and V. cholerae. The second part of this research was devoted to the investigation of gold MLTs as compared to other methods like vortex, sonication, and bead-beating as a platform for inactivating infectious pathogens. It was found that by utilizing gold MLTs, pathogenic bacteria was rendered inactive and thus safe for domestic and international shipping. The third and most compelling part of this work was dedicated to the elucidation of the non-thermal mechanism behind the gold MLTs. It was first noticed that DNA/RNA and proteins were being extracted at low microwave settings and subsequently being degraded at higher settings. Further investigation led to the discovery that as microwave conditions were held constant and thus temperature was constant, that genomic DNA and proteins were being degraded as oxygen content increased. This led to investigating reactive oxygen species (ROS). Specific fluorescent ROS probes were utilized to detect singlet oxygen, hydroxyl radicals, and superoxide anion radicals. It was observed that as oxygen concentration increased or microwave irradiation settings increased, there were more ROS generated with the use of gold MLTs; therefore, ROS serves as one part of the non-thermal mechanism. Finally, microwave effects on DNA/RNA nucleases structure and activity was investigated. It was found that with the use of gold MLTs, nucleases can be rendered inactive while still allowing for intracellular component detection.