Towards electrochemical diagnostics of biochemical free radical species in aqueous microliter volumes

Chainani, Edward

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https://hdl.handle.net/2142/42209 Copy

Description

Title

Towards electrochemical diagnostics of biochemical free radical species in aqueous microliter volumes

Author(s)

Chainani, Edward

Issue Date

2013-02-03T19:27:58Z

Director of Research (if dissertation) or Advisor (if thesis)

Scheeline, Alexander

Doctoral Committee Chair(s)

Scheeline, Alexander

Committee Member(s)

• Bailey, Ryan C.
• Pearlstein, Arne J.
• Wieckowski, Andrzej

Department of Study

Chemistry

Discipline

Chemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• acoustic levitation
• levitated drop
• microelectrode
• free radicals
• microfabrication
• lithography
• superoxide
• microfluidics
• electrochemistry
• spectroscopy

Abstract

Reactive oxygen species (ROS) are free radicals often implicated in disease states. In probing related enzyme processes, reactant consumption must be minimized. Levitated drops show potential as microreactors where radicals are present as reactants or products. Solid/liquid interfaces are absent or minimized, avoiding adsorption and interfacial reaction of conventional microfluidics. Probing small volumes electrochemically requires an integrated sensor with reference, counter-, and one or more working electrodes. Microfabrication allows production of planar gold sensor microelectrodes. A design that placed eight copies of the sensor on a single wafer maximized throughput and use of available substrate area. Titanium was found to be superior to chromium in corrosion resistance while providing adequate adhesion between gold and the polyimide-passivated silicon substrate. A lithographic process using image reversal with lift-off proved superior to etching in terms of line width range, allowing resolution of narrow electrodes (<25 μm) and wider conductive paths. Photosensitive polyimide proved capable of insulating non-electroactive sensor regions, prevented the occurrence of metal delamination encountered in previous electrode designs, as well as defining active electrode areas. Laser milling detached individual electrodes from the wafer and shaped the tip for piercing the levitated drop. The resulting three-electrode sensor with microdisk gold working electrode of radius 19 μm was characterized using ferrocenemethanol in aqueous buffer. Using cyclic voltammetry, the electrochemically active surface area was estimated by combining a recessed microdisk electrode model with the Randles-Sevcik equation. Computer-controlled ballistic introduction of reactant droplets into a levitated drop was developed. Chronoamperometric measurements of ferrocyanide with the microfabricated electrode demonstrated the electrochemical monitoring of reactions in a levitated drop, as well as the feasibility of adding reactants ballistically to the drop. Although concentration increases with time due to drop evaporation, it was found to be predictable with a linear evaporation model. Comparison of diffusion-limited currents in pendant and levitated drops shows that convection arising from acoustic levitation causes an enhancement of diffusion-limited current on the order of 16%. The study of ROS-mediated processes requires a supply of the ROS under investigation. A flow system was designed to generate superoxide radical anion (O2 -) at near-neutral pH by rapid mixing of potassium superoxide dissolved in dimethyl sulfoxide by two- stage mixing with aqueous buffers. This system’s intended application is the delivery of O2 - to bacteria immobilized on filters for investigating attenuation by O2 - on Salmonella enterica serovar typhimurium. Measurements of flow rates and calculations of residence times indicate final aqueous O2 - concentrations up to 50 μM are possible. A spectrophotometric flow cell was devised to confirm the concentration of superoxide.

Graduation Semester

2012-12

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Copyright and License Information

Copyright 2012 Edward T. Chainani

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