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A Novel Mass Spectrometry Method to Study Reaction Intermediates and Development of AuTeCDs for Scavenging ROS in Live Cells

Xu, Chang
http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1597326111937675

Abstract Details

2020, Doctor of Philosophy (PhD), Ohio University, Chemistry and Biochemistry (Arts and Sciences).
Mass Spectrometry (MS) has become a widely used technique for characterization and identification of both small and large molecules. This project presents a novel quantification approach, based on electrochemistry (EC) combined with mass spectrometry (MS), in which the oxidation current recorded from an electrochemistry (EC) measurement and the oxidation reaction yield from MS signal changes upon electrolysis can be used to identify chemicals. In combination with liquid chromatography (LC), the method can also be applied to complex mixture analysis. The striking advantage of such a method is that no standard compound is required. Several analyte molecules such as dopamine, norepinephrine, rutin, and glutathione were successfully identified using our method with the quantification error ranging from -3.4% to +4.6 %. Analytes in complex samples (e.g., uric acid in urine) were also accurately measured. In our experiments, a minimal amount of sample (e.g., 300 pmol of the compound) was used for quantification indicating our method is highly sensitive. The results presented here demonstrate that mass spectrometry is a powerful technique that not only can detect metabolites at low concentrations, but also can be used for capturing and characterizing the short-lived reaction intermediates due to its gentle ionization process which transfer products directly from liquid to the gas phase. By using both electrospray ionization (ESI)-MS and desorption electrospray ionization (DESI)-MS techniques, Pd (IV) and Au (III) intermediates were successfully captured and the reaction mechanism therefore was confirmed. The second study presented here is the development of novel nanoparticles to detect and scavenge ROS in living cells. Oxidative reactive oxygen species (ROS) are held in dynamic equilibrium with reducing species in living systems. When present at appropriate concentrations, ROS can have beneficial antimicrobial and anti-inflammatory effects. However, excessive ROS can oxidize lipids, proteins, nucleic acids, and other metabolites, putting immense stress on the biological environment in which they are contained. If left untreated, ROS can damage cell membranes, denature proteins, damage DNA, and lead to inflammation. Nature has evolved strategies for eliminating ROS in biological systems through the actions of enzymes such as catalase and superoxide dismutase that scavenge free radicals and protect the body from oxidative damage. Despite the efficacy of these enzymes, they are susceptible to inactivation and require specific physiological conditions. Therefore, there is considerable interest in developing a highly stable, artificial enzymes to augment the removal of ROS and protect the body from free radical damage. Over the past several years, nanomaterials with enzyme-like properties (nanozymes) have been gaining traction as potential alternatives for natural enzymes. However, developing highly active nanozymes with enhanced specificities remains a considerable challenge for biomimetic chemists. In this project, a novel superoxide dismutase mimetic nanozyme was developed using carbon dots (CD) and gold nanoparticles (Au) coupled to a tellurium (Te) active center. Te has been used previously as an active site mimetic of glutathione reductase and here, we used our Au-TeCD nanozymes to scavenge ROS in vitro and live cells. Notably, we were able to quantify ROS scavenging using a highly-sensitive dynamical fluorescence imaging analysis of superoxide anions in state of dynamic motion. Our results showed that our Au-TeCDs nanozymes can scavenge with high specificity and efficiency with a detection limit of 7.37 nM. We anticipate that these novel nanozymes will be useful for detecting high level of ROS in cells with compromised physiologies, including cancer and neurodegenerative cells.
Justin Holub (Advisor)
Peter Harrington (Committee Member)
Travis White (Committee Member)
Peter Jung (Committee Member)
144 p.
Analytical Chemistry; Biochemistry; Chemistry
Mass Spectrometry; DESI quantification; Electrochemistry; Capturing reaction intermediates; Nanozyme; ROS Scavenger

Recommended Citations

Citations

  • Xu, C. (2020). A Novel Mass Spectrometry Method to Study Reaction Intermediates and Development of AuTeCDs for Scavenging ROS in Live Cells [Doctoral dissertation, Ohio University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1597326111937675

    APA Style (7th edition)

  • Xu, Chang. A Novel Mass Spectrometry Method to Study Reaction Intermediates and Development of AuTeCDs for Scavenging ROS in Live Cells. 2020. Ohio University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1597326111937675.

    MLA Style (8th edition)

  • Xu, Chang. "A Novel Mass Spectrometry Method to Study Reaction Intermediates and Development of AuTeCDs for Scavenging ROS in Live Cells." Doctoral dissertation, Ohio University, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1597326111937675

    Chicago Manual of Style (17th edition)

ohiou1597326111937675
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This open access ETD is published by Ohio University and OhioLINK.