Publication date: 5th July 2021
Expression of the enzyme beta-galactosidase can be used as a marker for cancer cell senescence and thus drug effectiveness. MDA-MB-231 is an invasive breast cancer cell line which exhibits a high degree of phenotypic heterogeneity. Most cellular assays focus on aggregate statistics from large numbers of cells. Because cancer cells are heterogeneous with respect to enzyme activity, the development of treatments is difficult because bulk statistics mask the variability in the enzymatic activity of individual cells. In order to obtain a more accurate picture of enzyme activity, a method to measure the distribution of single-cell activities is needed.
We have developed a microfluidic device for analyzing beta-galactosidase expression in a relatively large number of MDA-MB-231 cells on a single-cell basis. The device uses an array of bipolar (floating) electrodes to capture cells by dielectrophoresis (DEP). DEP offers a label free method of manipulating cells with minimal impact on cellular function. Importantly, the fluorogenic beta-galactosidase assay composition was optimized for high activity in a low conductivity environment suitable for DEP. The captured cells are fluidically transferred and recaptured in reaction chambers and the fluorogenic assay buffer is added. An ionic liquid is used to isolate the cells and prevent cross-talk between chambers. Finally, a high AC voltage is used to lyse the cells and the enzyme activity is measured by fluorescence microscopy. Small scale implementation shows successful beta-galactosidase assay results from captured and isolated MDA cells.
The device developed can be used to characterize the variability of beta-galactosidase activity in MDA-MB-231 cells. Characterization of this enzymatic activity will facilitate studies of MDA cellular senescence following drug challenges and other stressors. Furthermore, the device is adaptable to other types of fluorometric assay such as other enzymes or nucleic acid amplification. The authors gratefully acknowledge funding from the NIH R21 Trailblazer Award.
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