This proposal describes research on a new microanalytical technique the investigators have termed """"""""electrophoretically mediated micro-analysis"""""""" (EMMA). EMMA is based on the use of differential electrophoretic mobility to mix nl volumes of reagents and carry out chemical reactions within small analyte zones in a capillary electrophoresis system. Reaction products are subsequently transported to absorbance or fluorescence detectors. Immunological assays, enzyme assays, and the determination of kinetic constants for enzymes will be examined with 0.01 - 10 nanoliter samples. Under most favorable conditions EMMA will be able to detect 100- 1000 molecules of an enzyme. This will allow enzymes to be detected and their kinetic constants determined from a single cell. The great advantage of this system is that it incorporates microsampling, high resolution capillary electrophoresis, highly specific enzyme reactions, and the sensitivity of modern electrophoresis detectors into a single electrophoretic system. The proposed research is divided into five activities; to 1) design and optimize sample inlet, reagent introduction, and surface pacivation techniques for the execution of EMMA, 2) develop theoretical models that will assist in optimizing the kinetics and selectivity of the system in the separation mode, 3) explore the utility of this system in enzyme assays that are diffusion controlled and kinetically optimized which monitor product formation with ultrahigh sensitivity laser induced fluorescence and bioluminescence based detection systems, 4) develop enzyme amplified immunological assays in capillary columns packed with microparticulate immunosorbents that are electrophoretically pumped, and 5) develop biosensors based on EMMA technology for the detection of both small molecules and enzymes in process streams.
| Patterson, D H; Harmon, B J; Regnier, F E (1996) Dynamic modeling of electrophoretically mediated microanalysis. J Chromatogr A 732:119-32 |
