Microfabricated flow chamber for fluorescence-based chemistries and stopped-flow injection cytometry.
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 (r2 = 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.
Hodder
P S
PS
Department of Chemistry, University of Washington, Seattle 98195, USA.
Blankenstein
G
G
Ruzicka
J
J
D003902:Detergents / Q000032:analysis; D005434:Flow Cytometry / Q000295:instrumentation*; D005470:Fluorometry / Q000295:instrumentation; D007700:Kinetics; D008856:Microscopy, Fluorescence; D012697:Serine Endopeptidases / Q000032:analysis
D003902:Detergents; D012697:Serine Endopeptidases; C032923:microbial serine proteinases