Elucidation of a non-thermal mechanism for DNA/RNA fragmentation and protein degradation when using Lyse-It


Autoři: Tonya M. Santaus aff001;  Ken Greenberg aff001;  Prabhdeep Suri aff001;  Chris D. Geddes aff001
Působiště autorů: Chemistry and Biochemistry Department, University of Maryland, Baltimore County, Baltimore, Maryland, United States of America aff001;  Institute of Fluorescence, University of Maryland, Baltimore County, Baltimore, Maryland, United States of America aff002
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article
doi: 10.1371/journal.pone.0225475

Souhrn

Rapid sample preparation is one of the leading bottlenecks to low-cost and efficient sample component detection. To overcome this setback, a technology known as Lyse-It has been developed to rapidly (less than 60 seconds) lyse Gram-positive and–negative bacteria alike, while simultaneously fragmenting DNA/RNA and proteins into tunable sizes. This technology has been used with a variety of organisms, but the underlying mechanism behind how the technology actually works to fragment DNA/RNA and proteins has hitherto been studied. It is generally understood how temperature affects cellular lysing, but for DNA/RNA and protein degradation, the temperature and amount of energy introduced by microwave irradiation of the sample, cannot explain the degradation of the biomolecules to the extent that was being observed. Thus, an investigation into the microwave generation of reactive oxygen species, in particular singlet oxygen, hydroxyl radicals, and superoxide anion radicals, was undertaken. Herein, we probe one aspect, the generation of reactive oxygen species (ROS), which is thought to contribute to a non-thermal mechanism behind biomolecule fragmentation with the Lyse-It technology. By utilizing off/on (Photoinduced electron transfer) PET fluorescent-based probes highly specific for reactive oxygen species, it was found that as oxygen concentration in the sample and/or microwave irradiation power increases, more reactive oxygen species are generated and ultimately, more oxidation and biomolecule fragmentation occurs within the microwave cavity.

Klíčová slova:

Beef – Fluorescence – Microwave radiation – Oxygen – Reactive oxygen species – Spinach – Proteolysis – Argon


Zdroje

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2019 Číslo 12