Microfabricated Flow Chamber for Fluorescence-based Chemistries and Stopped-flow Injection Cytometry

Abstract

A microfabricated flow chamber (MFC) suitable for performing liquid-based fluorimetric assays is introduced. Precision delivery of microliter volumes of sample and reagent to the MFC is accomplished by a double-syringe-pump flow injection analysis (FIA) apparatus. The FIA–MFC system also combines the ‘sheath flow’ technique (traditionally used in flow cytometry) and stopped-flow FIA as a way to allow sample and reagent streams to be mixed reproducibly. The applicability of this FIA–MFC system to bioanalytical assays is demonstrated by performing an enzymatic assay with an artificial fluorigenic substrate to determine the activity of Savinase, a proteolytic enzyme. When coupled to a fluorescence microscope platform, quantitative analysis of the reaction product is possible. Experiments showed that the FIA–MFC system was capable of performing the assay with good reproducibility of injection (1.5%), and linearity of response (r ² = 0.9997) in activity ranges of analytical interest. Owing to the incorporation of flow cytometry sheath flow principles into an FIA format, the FIA–MFC system is a suitable tool for cytometric studies.