Temporal Sampling of Enzymes from Live Cells by Localized Electroporation and Quantification of Activity by SAMDI Mass Spectrometry.
cell sampling
electroporation
enzyme activities
microfluidics
nanotechnologies
Journal
Small (Weinheim an der Bergstrasse, Germany)
ISSN: 1613-6829
Titre abrégé: Small
Pays: Germany
ID NLM: 101235338
Informations de publication
Date de publication:
07 2020
07 2020
Historique:
received:
29
01
2020
revised:
07
04
2020
accepted:
16
04
2020
pubmed:
27
5
2020
medline:
4
5
2021
entrez:
27
5
2020
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 32452612
doi: 10.1002/smll.202000584
pmc: PMC7401324
mid: NIHMS1611409
doi:
Substances chimiques
Enzymes
0
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, Non-P.H.S.
Langues
eng
Sous-ensembles de citation
IM
Pagination
e2000584Subventions
Organisme : NCI NIH HHS
ID : U54 CA199091
Pays : United States
Organisme : NIH HHS
ID : T32GM105538
Pays : United States
Organisme : NCI NIH HHS
ID : P30 CA060553
Pays : United States
Organisme : NIGMS NIH HHS
ID : R21 GM132709
Pays : United States
Organisme : NIH HHS
ID : U54CA199091
Pays : United States
Informations de copyright
© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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