Flow injection techniques for enzymatic and cellular drug discovery assays.

摘要

The research in this thesis develops novel Flow Injection (FI) techniques for fluorescence-based drug discovery assays in microfabricated and miniature structures. Automated instrumentation is constructed to perform quantitative biochemical and pharmacological assays on enzyme and whole cell samples. Data from the assays are analyzed to determine enzyme activity, as well as the potency and binding kinetics of muscarinic receptor agonists and antagonists.; A strategy for mixing in microfabricated structures is introduced. An instrument is developed that combines a FI apparatus with a microfabricated flow chamber (MFC) to demonstrate the utility of the mixing strategy. The FI-MFC instrument combines a "sheath flow" technique (traditionally used in flow cytometry) and stop-flow FI methodology to reproducibly mix sample and reagent streams in the MFC. An enzymatic assay with a fluorigenic substrate is executed to determine the activity of Savinase, a proteolytic enzyme. When coupled to a fluorescence microscope platform, quantitative, reproducible analysis of the enzymatic reaction product in activity ranges of analytical interest is demonstrated, proving that stop-flow FI techniques based on diffusion can be used as a reliable mixing method in micrometer dimensions.; A novel Flow Injection-Renewable Surface (FI-RS) technique is introduced for the execution of automated pharmacology-based assays with living cells. Cells are attached to microcarrier beads, which serve as the disposable and renewable surface on which the assay is performed. The FI apparatus allows reproducible and precise control of the concentration gradient of chosen muscarinic receptor agonists (carbachol, acetylcholine, pilocarpine) and antagonists (atropine, pirenzepine) delivered to cells trapped in a specially designed detector flow chamber. The RS methodology eliminates problems associated with diminishing biological response vis-a-vis traditional functional assays that are performed repetitively on the same group of cells.; Functional assays are performed with the FI-RS instrument using Chinese Hamster Ovary (CHO) cells transfected with the rat muscarinic receptor (ml) as the biological material. The intracellular calcium elevation resulting from the agonist-receptor interaction is measured via a calcium sensitive fluorescent probe (fura-2) and a fluorescence microscope photometry system. Using this technique, reproducible intracellular calcium responses are measured. Equilibrium and kinetic pharmacological parameters are quantified that compare favorably to literature values.