Temporal Sampling of Enzymes from Live Cells by Localized Electroporation and Quantification of Activity by SAMDI Mass Spectrometry
| Autor: | Milan Mrksich, S. Shiva P. Nathamgari, Horacio D. Espinosa, Eric J. Berns, Elamar Hakim Moully, Prithvijit Mukherjee, Cesar A. Patino, John A. Kessler, Lingqian Chang |
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| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
Cells
Microfluidics Phosphatase Synthetic membrane 02 engineering and technology 010402 general chemistry Mass spectrometry 01 natural sciences Article Time Biomaterials Cell membrane Cell Line Tumor medicine Humans General Materials Science Enzyme Assays chemistry.chemical_classification Electroporation General Chemistry 021001 nanoscience & nanotechnology 0104 chemical sciences Enzymes Enzyme medicine.anatomical_structure chemistry Cytoplasm Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Biophysics 0210 nano-technology Biotechnology |
| Zdroj: | Small |
| Popis: | Measuring changes in enzymatic activity over time from small numbers of cells remains a significant technical challenge. In this work, a method for sampling the cytoplasm of cells is introduced to extract enzymes and measure their activity at multiple time points. A microfluidic device, termed the live cell analysis device (LCAD), is designed, where cells are cultured in microwell arrays fabricated on polymer membranes containing nanochannels. Localized electroporation of the cells opens transient pores in the cell membrane at the interface with the nanochannels, enabling extraction of enzymes into nanoliter-volume chambers. In the extraction chambers, the enzymes modify immobilized substrates, and their activity is quantified by self-assembled monolayers for matrix-assisted laser desorption/ionization (SAMDI) mass spectrometry. By employing the LCAD-SAMDI platform, protein delivery into cells is demonstrated. Next, it is shown that enzymes can be extracted, and their activity measured without a loss in viability. Lastly, cells are sampled at multiple time points to study changes in phosphatase activity in response to oxidation by hydrogen peroxide. With this unique sampling device and label-free assay format, the LCAD with SAMDI enables a powerful new method for monitoring the dynamics of cellular activity from small populations of cells. |
| Databáze: | OpenAIRE |
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