Analysis of biochemical activity in small volumes (e.g. 10 to the -6 ml) allows measurement of the activity of individual enzymes and of the biochemical activity of individual cells or of microcolonies. We propose to investigate a method for making such analyses rapidly, by use of small gel or liquid droplets (about 100 micron diameter) in combination with flow cytometry (microdroplet/flow cytometry approach). The droplets will be statistically occupied, such that an individual droplet has a high probability of containing 0 or 1 active entity (enzyme molecule or cell), and a low probability of containing 2 or more entities (a multiply occupied droplet). A macroscopic liquid specimen can be rapidly converted into a large population of droplets. By providing suitable substrates and cofactors, or the nutrients and reagents for an extracellular biochemical assay, the accumulation of a fluorescent product can occur and be confined within the droplet. A large number of biochemical systems of importance to fundamental biological problems and to problems in medicine should be amenable to this method. We propose to investigate the basic properties of this small volume element method, and then to use it in the study of several systems. An important problem in enzymology relates to the mechanisms in changes of enzymatic activity, particularly inactivation. By measuring the activity of a large number (e.g. 10 to the 4) of individual enzyme molecules, it will be possible to readily study whether inactivation is an """"""""all or nothing"""""""" process for individual enzyme molecules, using several different enzymes. Microanalysis within small volume elements is also applicable to biochemical activity measurements on individual cells, including bacteria, actinomyces, yeast, molds, and mammalian cells. This technique opens up the possibility of radically better strain improvement by the examination of the capability of individual cells or microcolonies to produce important biochemicals. By rapidly obtaining many individual measurements of the biochemical activity within droplets, it should be possible to readily compute a population distribution based on specific biochemical activity. Such a distribution could form the basis for population studies, or for screening for specific biochemical activities.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM034077-02
Application #
3284533
Study Section
(SSS)
Project Start
1984-08-01
Project End
1987-07-31
Budget Start
1985-08-01
Budget End
1986-07-31
Support Year
2
Fiscal Year
1985
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