Temporal Sampling of Enzymes from Live Cells by Localized Electroporation and Quantification of Activity by SAMDI Mass Spectrometry

Overview

Journal Small

Date 2020 May 27

PMID 32452612

Citations 9

Authors

Prithvijit Mukherjee

Eric J Berns

Cesar A Patino

Elamar Hakim Moully

Lingqian Chang

S Shiva P Nathamgari

John A Kessler

Milan Mrksich

Horacio D Espinosa

Affiliations

Soon will be listed here.

Abstract

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.

Citing Articles

Well Plate-Based Localized Electroporation Workflow for Rapid Optimization of Intracellular Delivery.

Patino C, Sarikaya S, Mukherjee P, Pathak N, Espinosa H Bio Protoc. 2024; 14(14):e5037.

PMID: 39100599 PMC: 11291937. DOI: 10.21769/BioProtoc.5037.

Analysis of In Situ Electroporation Utilizing Induced Electric Field at a Wireless Janus Microelectrode.

Sun H, Yu L, Chen Y, Yang H, Sun L Micromachines (Basel). 2024; 15(7).

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Transepithelial Electrical Impedance Increase Following Porous Substrate Electroporation Enables Label-Free Delivery.

Brooks J, Heiman T, Lorenzen S, Mungloo I, Mirfendereski S, Park J Small. 2024; 20(25):e2310221.

PMID: 38396158 PMC: 11186731. DOI: 10.1002/smll.202310221.

Single cell transcriptomics reveals reduced stress response in stem cells manipulated using localized electric fields.

Mukherjee P, Peng C, McGuire T, Hwang J, Puritz C, Pathak N Mater Today Bio. 2023; 19:100601.

PMID: 37063248 PMC: 10102005. DOI: 10.1016/j.mtbio.2023.100601.

Biological mass spectrometry enables spatiotemporal 'omics: From tissues to cells to organelles.

Pade L, Stepler K, Portero E, DeLaney K, Nemes P Mass Spectrom Rev. 2023; 43(1):106-138.

PMID: 36647247 PMC: 10668589. DOI: 10.1002/mas.21824.

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