University of California

Gavin D Meredith

Bruce J Tromberg

Nancy L Allbritton

Christopher E Sims

Michael W Berns

Tatiana B Krasieva

Chao L Lee

The activity of intracellular chemical reactions of molecules is measured by the use of fluorescently labeled substrate molecules that undergo a change in electrophoretic mobility upon chemical reaction such as that catalyzed by an enzyme. Specificity is achieved by using labeled substrate molecules that can be acted upon only by specific enzymes. Thus the activity of a specific enzyme or class of enzymes can be determined. Measurements are made with the intracellular presence of such substrate molecules, at some time of interest, typically after exposure of the cell to a stimulus that activates a particular enzymatic pathway. To ensure accuracy, measurements must be made in a timely manner so as to minimize chemical reactions occurring subsequent to the time of interest. Fast controllable laser lysis is used to obtain the contents of a single cell into which reporter substrate molecules have been introduced. The cell contents are then subjected to capillary electrophoresis and enzymatic activity is determined by comparing amounts of substrate molecules to amounts of enzymatically altered substrate molecules which are separated by the electrophoresis and identified by the presence of a fluorescent label.

Method and apparatus for detecting enzymatic activity using molecules that change electrophoretic mobility

Fast lysis of a single cell or cellular component thereof is performed by generating a shock wave in a medium in which the cell or cellular component thereof is positioned. The cell or cellular component thereof is either positioned by laser tweezers or cultured as an adhered cell or cellular component thereof to minimize manipulation trauma. The disclosed method completely lyses a single cell or cellular component thereof in a controllable manner in milliseconds or less followed immediately by the loading of the cellular contents into a capillary for analyte separation and detection. The cell or cellular component thereof is adjacent the inlet of an electrophoretic column through which a gravity siphon flow of the medium is maintained. The lysed contents of the cell or cellular component thereof enter the electrophoretic column in less than 33 msec, are separated and analyzed by laser induced fluorescence. The method takes advantage of the shock wave produced by a highly focused laser pulse which is created in a medium adjacent to the cell or cellular component thereof. In the illustrated embodiment the laser pulse is focused in the glass substrate at or near a glass-to-buffer interface of a cell chamber in which the cell or cellular component thereof to be lysed has been cultured.

Fast controllable laser lysis of cells for analysis

Growing community of inventors

Cardiff-by-the-Sea, CA, United States of America

Gavin D Meredith