Development and Characterization of a Stopped-Flow-Bypass Analysis System With Applications To Biochemical Measurements

Abstract

A new apparatus called Bypass Trapped Flow Analysis System (ByT-FAS) is described. A properly designed ByT-FAS gives an analyst the ability to use analyte sample volumes of 10 to 200 μL [or more] and reagent volumes of approximately the same size. The sample and reagent are injected into their respective carrier streams and attain physical steady state concentrations in the detection cell within approximately 15 to 45 seconds after injection. Upon achievement of simultaneous sample and reagent physical steady state concentrations, the system flow is diverted around the detection cell and the reaction mixture is trapped in the detection cell. The concentration of the sample and reagent in the detection cell can be readily computed from knowledge of the original concentrations of the sample and reagent and the flow rates of the streams propelling the sample and reagent. ByT-FAS was demonstrated to be useful for direct measurements of analytes in liquid solutions and for assays which utilize equilibrium and/or kinetic methods to create measurable product(s) for ultraviolet/visible spectrophotometry, fluorimetry, and chemiluminescence. Enzyme activities and fundamental enzyme kinetic parameters (Kms, Kis, VMAXs) were determined directly. Genetic transcription levels of luciferase in whole intact E. coli cells were also determined using chemiluminescent detection. Flow system configuration, components, and flow ratios were investigated for their effects on achieving physical steady state signals in the detector. It is believed that this new type of instrumentation will be of significant use for the analytical chemical, biochemical, molecular biology, biotechnology, environmental, pharmaceutical and medical communities for those measurements which require direct knowledge of the concentration of the reactants and products during quantitation.