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
prolekare.web.journal.doi_sk: 10.1371/journal.pone.0225475


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:

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


1. Aslan K, Previte MJR, Zhang Y, Gallagher T, Baillie L, Geddes CD. Extraction and detection of DNA from Bacillus anthracis spores and the vegetative cells within 1 min. Analytical Chemistry. 2008;80(11):4125–32. doi: 10.1021/ac800519r 18459738.

2. Aslan K, Zhang Y, Hibbs S, Baillie L, Previte MJR, Geddes CD. Microwave-accelerated metal-enhanced fluorescence: application to detection of genomic and exosporium anthrax DNA in <30 seconds. The Analyst. 2007;132(11):1130–8. doi: 10.1039/b707876e 17955147.

3. Dragan AI, Albrecht MT, Pavlovic R, Keane-Myers AM, Geddes CD. Ultra-fast pg/ml anthrax toxin (protective antigen) detection assay based on microwave-accelerated metal-enhanced fluorescence. Analytical Biochemistry. 2012;425:54–61. doi: 10.1016/j.ab.2012.02.040 22406431

4. Melendez JH, Huppert JS, Jett-Goheen M, Hesse EA, Quinn N, Gaydos CA, et al. Blind evaluation of the microwave-accelerated metal-enhanced fluorescence ultrarapid and sensitive Chlamydia trachomatis test by use of clinical samples. Journal Of Clinical Microbiology. 2013;51(9):2913–20. doi: 10.1128/JCM.00980-13 23804384.

5. Melendez JH, Santaus TM, Brinsley G, Kiang D, Mali B, Hardick J, et al. Microwave-accelerated method for ultra-rapid extraction of Neisseria gonorrhoeae DNA for downstream detection. Analytical Biochemistry. 2016;510:33–40. doi: 10.1016/j.ab.2016.06.017 27325503

6. Santaus TM, Melendez JH, Negesse MY, Harvery A, Cyr M, Ladd P, et al. Lyse-It™: A Rapid Platform for Cellular Lysing and Tunable DNA/Protein Fragmentation. In: Geddes CD, editor. Microwave Effects on DNA and Proteins: Springer; 2017. p. 275–96.

7. Zhang Y, Agreda P, Kelley S, Gaydos C, Geddes CD. Development of a Microwave—Accelerated Metal-Enhanced Fluorescence 40 Second, <100 cfu/mL Point of Care Assay for the Detection of Chlamydia Trachomatis. IEEE Transactions on Biomedical Engineering. 2011;58(3):781. doi: 10.1109/TBME.2010.2066275 20709634

8. Aslan K, Geddes CD. Microwave-Accelerated and Metal-Enhanced Fluorescence Myoglobin Detection on Silvered Surfaces: Potential Application to Myocardial Infarction Diagnosis. Plasmonics (Norwell, Mass). 2006;1(1):53–9. doi: 10.1007/s11468-006-9006-7 19444320.

9. Aslan K, Geddes CD. Microwave-accelerated metal-enhanced fluorescence: platform technology for ultrafast and ultrabright assays. Analytical Chemistry. 2005;77(24):8057–67. doi: 10.1021/ac0516077 16351156.

10. Previte MJR, Geddes CD. Microwave-triggered chemiluminescence with planar geometrical aluminum substrates: theory, simulation and experiment. Journal of Fluorescence. 2007;17(3):279–87. doi: 10.1007/s10895-007-0170-8 17404821. CODEN: JOFLEN. Digital Object Identifier: 10.1007/s10895-007-0170-8. ISSN: 1053-0509 (print). Key Phrase Headings: microwave-triggered chemiluminescence.

11. Kadir A, Chris DG. New tools for rapid clinical and bioagent diagnostics: microwaves and plasmonic nanostructures. Analyst. 2008;133(11):1469. doi: 10.1039/b808292h 18936822

12. Previte MJR, Asian K, Geddes CD. Spatial and Temporal Control of Microwave Triggered Chemiluminescence: A Protein Detection Platform. Analytical Chemistry. 2007;79(18):7042–52. doi: 10.1021/ac071042+ 17696497.

13. Santaus TM, Li S, Ladd P, Harvey A, Cole S, Stine OC, et al. Rapid sample preparation with Lyse-It(R) for Listeria monocytogenes and Vibrio cholerae. PLoS One. 2018;13(7):e0201070. Epub 2018/07/26. doi: 10.1371/journal.pone.0201070 30044836 a patent related to microwave-based lysing and DNA fragmentation ("Assays for pathogen detection using microwaves for lysing and accelerating metal-enhanced fluorescence", US9500590B2). Those patents are licensed to Lyse-It LLC, a Maryland-based biotechnology company, which Professor Geddes currently holds stock in. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

14. Santaus T. Focused microwaves for cellular lysing, DNA/RNA and protein/enzyme fragmentation. In preparation2019.

15. Krumova K, Cosa G. Overview of Reactive Oxygen Species. 2016:3.

16. Tripathy S, Mohanty PK. Reactive Oxygen Species (ROS) are Boon or Bane. International Journal of Pharmaceutical Sciences and Research. 2017;8(1):1–16.

17. Santaus T, Zhang F, Li S, Stine OC, Geddes CD. Effects of Lyse-It®on Endonuclease fragmentation, function and activity. In Preparation2019.

18. Lunec J, Holloway KA, Cooke MS, Faux S, Griffiths HR, Evans MD. Urinary 8-oxo-2'-deoxyguanosine: redox regulation of DNA repair in vivo? Free Radical Biology & Medicine. 2002;33(7):875–85. doi: 10.1016/s0891-5849(02)00882-1 12361799.

19. Ray RS, Mujtaba SF, Dwivedi A, Yadav N, Verma A, Kushwaha HN, et al. Singlet oxygen mediated DNA damage induced phototoxicity by ketoprofen resulting in mitochondrial depolarization and lysosomal destabilization. Toxicology. 2013;314:229–37. doi: 10.1016/j.tox.2013.10.002 24128752

20. Kristine MR, Michael SJ, Mariana P, Jeffrey SM, Meredith FR, Tory MH, et al. Selective Fluorescent Imaging of Superoxide in vivo Using Ethidium-Based Probes. Proceedings of the National Academy of Sciences of the United States of America. 2006;(41):15038. doi: 10.1073/pnas.0601945103 17015830

21. Setsukinai K-i, Urano Y, Kakinuma K, Majima HJ, Nagano T. Development of novel fluorescence probes that can reliably detect reactive oxygen species and distinguish specific species. The Journal Of Biological Chemistry. 2003;278(5):3170–5. doi: 10.1074/jbc.M209264200 12419811.

22. Wardman P. Review Article: Fluorescent and luminescent probes for measurement of oxidative and nitrosative species in cells and tissues: Progress, pitfalls, and prospects. Free Radical Biology and Medicine. 2007;43:995–1022. doi: 10.1016/j.freeradbiomed.2007.06.026 17761297

23. Kim S, Fujitsuka M, Majima T. Photochemistry of Singlet Oxygen Sensor Green. 2013:13985.

24. Ragàs X, Jiménez-Banzo A, Sánchez-García D, Batllori X, Nonell S. Singlet oxygen photosensitisation by the fluorescent probe Singlet Oxygen Sensor Green. Chemical Communications (Cambridge, England). 2009;(20):2920–2. doi: 10.1039/b822776d 19436910.

25. Sugiyama I, Kojima S, Oku N, Sadzuka Y. Liposomalization of hydroxyphenyl fluorescein as a reagent for detecting highly reactive oxygen species. Colloid & Polymer Science. 2010;288(12/13):1293–300. doi: 10.1007/s00396-010-2256-0

26. Garcia-Diaz M, Huang Y-Y, Hamblin MR. Use of fluorescent probes for ROS to tease apart Type I and Type II photochemical pathways in photodynamic therapy. Methods. 2016;109:158–66. doi: 10.1016/j.ymeth.2016.06.025 27374076

27. Pannunzio NR, Lieber MR. AID and Reactive Oxygen Species Can Induce DNA Breaks within Human Chromosomal Translocation Fragile Zones. Molecular Cell. 2017;68(5):901–12. doi: 10.1016/j.molcel.2017.11.011 29220655

28. Waris G, Alam K. Attenuated antigenicity of ribonucleoproteins modified by reactive oxygen species. 1998:33.

29. Knight JA. Diseases related to oxygen-derived free radicals. Annals Of Clinical And Laboratory Science. 1995;25(2):111–21. 7785961.

30. Breen AP, Murphy JA. Reactions of oxyl radicals with DNA. Free Radical Biology & Medicine. 1995;18(6):1033–77. doi: 10.1016/0891-5849(94)00209-3 7628729.

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2019 Číslo 12