Nanobiosensors for oxidative stress and their applications

Open Access
Author:
Kim, Se-Hwa
Graduate Program:
Integrative Biosciences
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
February 26, 2007
Committee Members:
  • Michael V Pishko, Committee Chair
  • Cara Lynne Schengrund, Committee Member
  • Ahmed A Heikal, Committee Member
  • Peter J Butler, Committee Member
  • Peter John Hudson, Committee Member
Keywords:
  • biosensor
  • cell-based
  • enzyme
  • polymer
  • oxidative stress
Abstract:
Many chronic diseases, including vascular diseases, cancer, and neural degenerations, are accompanied by oxidative stress, an imbalance between pro-oxidants and anti-oxidants. Reactive oxygen species (ROS) as a pro-oxidant deeply involves in many cellular metabolisms which use redox-balance. For this reason, oxidative stress has been considered as one of the biomarkers for the abnormality of body conditions. Therefore, the various measurement systems of oxidative stress have been explored, for example, a measurement of oxidized biomolecules. However, there are a few methods to detect oxidative stress directly, due to their unstable properties. The precise methodology such as a biosensor to detect ROS directly is desirable for diagnostic purposes. In this dissertation, various biosensors for the measurement of intracellular oxidative stress are developed, including the fabrication, characterization, and cellular applications. In order to develop a biosensor for the measurement of intracellular oxidative stress, there are several requirements. First, the biosensor for intracellular measurements should be on the nano-scale for effective internalization with minimized the cellular side effects. Second, the materials used in the biosensor should be biocompatible and suitable for the easy transfer of analytes. To satisfy these requirements, poly(ethylene) glycol hydrogels were used for the biosensors for measurements of oxidative stress, hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) and superoxide anions (O<sub>2</sub><sup>•-</sup>). The first biosensor for the detection of H<sub>2</sub>O<sub>2</sub> was developed by a novel technique to encapsulate enzymes in PEG hydrogel without loss of enzymatic activity. Encapsulated horseradish peroxidase (HRP) can be used for effective conversion of H<sub>2</sub>O<sub>2</sub> to fluorescent material for the optical detection. The HRP-loaded nanobiosensor then was characterized and applied in macrophages phagocytic system. For the detection of superoxide anions (O<sub>2</sub><sup>•-</sup>) as another potent intracellular oxidative stress, superoxide dismutase (SOD) was integrated in the previous nanobiosensor. The fabricated SOD-HRP encapsulated nanobiosensor was then characterized by several methods, including the enzymatic activities by using superoxide anions generation system. Furthermore, the surfaces of nanospheres were modified by phospholipids for the internalization in non-phagocytic cells. These method allowed to detect O<sub>2</sub><sup>•-</sup>in smooth muscle cells and brain cancer cells. Finally, resultant cellular responses by oxidative stress were studied by fluorescent dye doped silica nanoparticles with bio-functional surface. One of the cellular responses mediated by oxidative stress is the migration of smooth muscle cells, involved in vascular disease and vessel remodeling. The migration of smooth muscle cells can be indicated by internalized fluorescent nanoparticles through membrane-type matrix metalloproteinase. Furthermore, the cellular responses to internalized nanoparticles were extensively studied, regarding to cytoskeletal structure. Therefore, this dissertation describes the fabrication, characterization, and applications of nanobiosensors for intracellular oxidative stress measurement and resultant cellular responses mediated by oxidative stress. These results in this thesis might contribute the field of nanobiosensors for the detection of intracellular analytes and analysis of cellular responses.