Micrototal Analysis System For Enzymatic Drug Metabolism and Analysis

Open Access
Author:
Hayes, Daniel James
Graduate Program:
Engineering Science and Mechanics
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
January 26, 2004
Committee Members:
  • Sabih I Hayek, Committee Member
  • Osama O Awadelkarim, Committee Member
  • Gregory S Mc Carty, Committee Member
  • Seong H Kim, Committee Member
  • Stephen Joseph Fonash, Committee Chair
Keywords:
  • Cell Based Biosensor
  • Microfluidics
  • metabolism
  • drug discovery
  • mass spectrometry
Abstract:
The goal of this thesis was to develop a laser based, matrix free, desorption and ionization mass spectrometry technique in a microscale array format that would have immediate application for drug and drug metabolite studies. This study also had a broader overriding goal contributing to the body of knowledge of cell patterning and microfluidic systems on a chip research. The research explored novel cell patterning techniques and biochemical sensing devices utilizing organic and inorganic low temperature deposited thin films. Specifically, this research sought to fabricate a microscale, cell based, device for drug metabolism testing using novel deposited thin films. This device was designed to identify and quantify drugs and associated metabolite products. A system utilizing liver derived microsomes and photocatalyzed PEG hydrogels can be used to pattern functional cytochrome p450 enzymes in situ, in PDMS microfluidic devices. This microfluidics system can then be used to biotransform drug molecules by enzymatic reaction. The metabolism products of the reaction can be identified and quantified by direct LDI-MS from the surface of chip. This study has shown the feasibility of conducting drug metabolism reactions and high speed LDI-MS analysis in a high throughput, PDMS chip based format. LDI-MS is a sensitive and rapid screening method which can analyze compounds in seconds compared to minutes for the state of the art electrospray ionization techniques. A microscale based system offers the advantages of the reduction of: costly sample consumption, waste by products and cross contamination issues. This technology coupled to a low cost, high speed, parallel device array platform can make a near term impact on the drug discovery industry by providing high content data on drug behavior and structure at early stages of the drug development cycle.