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

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 MALDI (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 This article is protected by copyright. All rights reserved. 3 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 labelfree assay format, the LCAD with SAMDI enables a powerful new method for monitoring the dynamics of cellular activity from small populations of cells.

High Throughput and Highly Controllable Methods for In Vitro Intracellular Delivery

“…Porous substrate electroporation is a particularly promising and under investigated engineered substrate method which we reviewed in detail. [155] -2000 µg mL −1 [154] DMEM, [154] PBS [155] [ 154,155] PI 0.668 0.6 nm r h + 2 [153] -20 [152] µg mL −1 , 100 µM [167] PBS [153] , high glucose DMEM [167] [ 152,153,159,160,163,165,167] Oligonucleotide Hypo-osmolar buffer, [152] Iso-osmolar buffer, [152] PBS [158] [ Plasmid DNA CS1-CAR 9 kb - [165] GFP ≈2000 3.3 kb -0.2 [151] -1000 [153] µg mL −1 DMEM [153] [ 151,153,159] gWiz GFP ≈3500 5.8 kb -<50 µg mL −1 [156] gWiz SEAP ≈4000 6.6 kb -5 [167] -100 [157] µg mL −1 High glucose DMEM [157,167] [ 156,157,167] mCherry ≈2400 4 kb -20 µg mL −1 [152] NF2 CRISPR/ Cas9 KO >5500 >9 kb - [168] OSKM pCAG ≈7900 13 kb - [160] pDsRed-C1 ≈2900 4.7 kb -100 µg mL −1 [154] pmaxGFP ≈2100 3.5 kb -5 µg mL −1 [167] High Glucose DMEM [167] [ 160,<...>…”

“…The last category of high throughput, high control systems, porous substrate systems ( Figure 2H) are substrates containing numerous micro-and nanopores on which cells are seeded and electroporated. Porous substrates can consist of commercially available membranes with random pore distribution, [151][152][153][154][155][156][157][158][159] or uniform arrays of pores on silicon chips. [160][161][162][163][164][165][166][167][168][169][170] Like nanostructures, cells seal around the pores which limits the electric field exposure to discrete regions of each cell.…”

“…Pulse frequencies have been reported from 1-200 Hz. The number of pulses applied per train range from 1-2400 pulses, while the number of trains is generally one and HeLa cervical epithelial Poly-L-Lysine [151] or Fibronectin [151,154] >95% [151] >80% [151] [ 151,153,154,159] HL-60 Promyeloblast, suspended >90% 65% a) [155] HT1080 Connective tissue Fibronectin [152] 50% [153] [ 152,153,159] Jurkat T lymphocyte, suspended Poly-L-Lysine or Fibronectin, [151] PEG-Silane [163] >95% [151] 75% [151] [ 151,163] KG1a Promyeloblast, suspended PEG-Silane 96% [163] K562 Lymphoblast, suspended PEG-Silane [163] 92% [163] 83.4% [163] [ 163,164] MDA-MB231 Mammary epithelial Fibronectin >99% [152] 70% [152] [ 152,158] NK-92 Natural killer, suspended 90% 74% [165] Mouse NIH3T3 Embryonic fibroblast Poly-l-lysine or fibronectin [151] >95% [151] 75% [151] […”

Microfluidic Surgery in Single Cells and Multicellular Systems

“…Instead of having nanotubes piercing into the cells, devices called the live cell analysis device (LCAD) used nanochannels (radius ~250 nm) formed by a nanoporous polycarbonate membrane, on which cells were cultured. 302,303 Similar to nanostraws, LCAD operated on the principle of membrane permeabilization using electroporation followed by an outflow of cytosol primarily by diffusion. The LCAD was sandwiched between two indium tin oxide electrodes.…”

Electrochemically and Mechanically Regulated Liquid Metal Gate via Giant Surface Tension Alteration

“…[ 6 ] In the field of bioengineering, to deliver DNAs or drugs into the cells, electroporation is an effective way to open holes on the cell membrane, basically made up of phospholipid. [ 7 ] The cell membrane can be self‐healing to seal the holes owning to the good fluidity of the phospholipid membrane. [ 8 ] Inspired by flowing cell membrane, liquid gate with electrical control is emerging to the surface.…”