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.