Donut-shaped chambers for analysis of biochemical processes at the cellular and subcellular levels.

Abstract

In order to study cell-cell variation with respect to enzymatic activity, individual live cell analysis should be complemented by measurement of single cell content in a biomimetic environment on a cellular scale arrangement. This is a challenging endeavor due to the small volume of a single cell, the low number of target molecules and cell motility. Micro-arrayed donut-shaped chambers (DSCs) of femtoliter (fL), picoliter (pL), and nanoliter (nL) volumes have been developed and produced for the analysis of biochemical reaction at the molecular, cellular and multicellular levels, respectively. DSCs are micro-arrayed, miniature vessels, in which each chamber acts as an individual isolated reaction compartment. Individual live cells can settle in the pL and nL DSCs, share the same space and be monitored under the microscope in a noninvasive, time-resolved manner. Following cell lysis and chamber sealing, invasive kinetic measurement based on cell content is achieved for the same individual cells. The fL chambers are used for the analysis of the same enzyme reaction at the molecular level. The various DSCs were used in this proof-of-principle work to analyze the reaction of intracellular esterase in both primary and cell line immune cell populations. These unique DSC arrays are easy to manufacture and offer an inexpensive and simple operating system for biochemical reaction measurement of numerous single cells used in various practical applications.