Multiple Sampling in Single-Cell Enzyme Assays Using CE-Laser-Induced Fluorescence to Monitor Reaction Progress

摘要

A novel method for assaying enzymes from a single cell or small cell populations is described. The key advantageof this method is the ability to repeatedly sample a single cell enzyme reaction. Whereas multiple sampling has beenachieved for larger cell tyes with a diameter of 1 mm, we report a technique by which single cell enzyme assaysof small cells (15 mum in diameter) can be repeatedly carried out. Individual cells were isolated using an inhouse-built micromanipulator and placed in nanoliterscale reaction vessels. The cells were lysed with solutioncontaining substrate, and enzyme activity was assayed by removing 5-nL aliquots with a recently developed nanopipettor. The reaction aliquot was then analyzed using capillary electrophoresis with laser-induced fluorescencedetection to quantitate enzyme activity. Sf9 cells were assayed at the single cell level and found te be highlybeterogeneous with respect to a-glucosidase II activity. Since only 5 nL of the single cell reaction was removed,multiple sampling was possible, allowing triplicate analysis of enzyme activity for each individual cell. Multiplesampling also permitted a single cell reaction to be monitored over time. The sensitivity of this method wasdemonstrated in the analysis of a low-abundance enzyme, al, 3-N-acetylgalactosaminyltransferase, from single HT29cells. Detecting the product of this enzyme reaction required minimzing the dilution of cellular contents. Todemonstrate the potential applications of this methodology in small scale biochemical analyses, single Arabidopsisknf embryos lacking the a-glucosidase I encoding KNOPF gene were assayed. Mutant embryos demonstrated insignificant conversion of a triglucose substrate, as compared to wild type, confirming the deletion of a-glucosidase I.Embryos were simultaneously assayed for a second enzyme, beta-galactosidase, illustrating that the mutants wereviable except for their lack of a-glucosidase I activity.