We propose continuation of a basic investigation using gel microdroplets (GMDs; e.g. 10 to 100 mu agarose particles) to analyze biochemical activity in very small volumes (e.g. 10-10 to 10-6 ml). We seek rapid: (1) determination of activity of individual enzyme molecules, cells (or microcolonies), (2) sorting of GMDs with cells of unusual activity, and (3) determination of growth of individual cells (or microcolonies) by using flow cytometry with either aqueous or non-aqueous fluidics. The GMDs are created rapidly and statistically occupied, such that an individual GMD has a high probability of initially containing 0 or 1 active entity (enzyme molecule or cell), and a low probability of initially containing 2 or more entities (a multiply occupied GMD). By providing suitable nutrients and reagents for a extracellular biochemical assay, fluorescent product is accumulated within individual GMDs. Alternatively, initial cells grow to form microcolonies of two or more cells, which are fluorescence labeled in proportion to the amount of specific cellular constituents. Many biochemical systems of importance to fundamental biological and medical problems appear amenable to this method. We propose continued investigation of the basic properties of this small volume method, and its use in the study of several fundamental problems. An important problem in enzymology relates to the mechanisms of changes in enzymatic activity, particularly inactivation. By measuring the activity of a large number (e.g. 104) of individual enzyme molecules, it will be possible to readily study whether inactivation is an """"""""all or nothing"""""""" or a gradual process for individual enzyme molecules, using several different enzymes. Microanalysis within the small volumes of GMDs is also applicable to biochemical activity measurements of individual microorganisms and mammalian cells, and is applicable to partially automated strain improvement through rapid sorting of GMDs with individual cells or microcolonies which have unusual activity. by rapidly obtaining many individual measurements of the biochemical activity within GMDs, it is possible to readily compute a population distribution based on specific biochemical activity. Such a distribution can form the basis for population studies, or for screening for sorting cells for specific biochemical activities. Rapid determinations of microbial activities and growth based on individual cells (or microcolonies) are potentially applicable to important clinical tests and environmental health problems.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM034077-04
Application #
3284530
Study Section
(SSS)
Project Start
1984-08-01
Project End
1990-07-31
Budget Start
1987-08-01
Budget End
1988-07-31
Support Year
4
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Massachusetts Institute of Technology
Department
Type
Schools of Arts and Sciences
DUNS #
City
Cambridge
State
MA
Country
United States
Zip Code
02139