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

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Temporal Sampling of Enzymes from Live Cells by Localized Electroporation and Quantification of Activity by SAMDI Mass Spectrometry.
Small ( IF 13.0 ) Pub Date : 2020-05-26 , DOI: 10.1002/smll.202000584
Prithvijit Mukherjee _{1,

2} , Eric J Berns ₃ , Cesar A Patino ₁ , Elamar Hakim Moully ₄ , Lingqian Chang ₁ , S Shiva P Nathamgari _{1,

2} , John A Kessler ₅ , Milan Mrksich _{3,

4,

6} , Horacio D Espinosa _{1,

2}

Affiliation

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.

中文翻译：

通过局部电穿孔对活细胞中的酶进行时间采样并通过 SAMDI 质谱法进行活性定量。

测量少量细胞的酶活性随时间的变化仍然是一项重大的技术挑战。在这项工作中，引入了一种对细胞的细胞质进行采样的方法来提取酶并在多个时间点测量其活性。设计了一种称为活细胞分析装置（LCAD）的微流体装置，其中细胞在含有纳米通道的聚合物膜上制造的微孔阵列中培养。细胞的局部电穿孔在细胞膜与纳米通道的界面处打开瞬时孔，从而能够将酶提取到纳升体积的室中。在提取室中，酶会修饰固定的底物，并通过自组装单分子层进行基质辅助激光解吸/电离 (SAMDI) 质谱分析来量化其活性。通过使用 LCAD-SAMDI 平台，演示了蛋白质向细胞的递送。接下来，研究表明可以在不损失活力的情况下提取酶并测量其活性。最后，在多个时间点对细胞进行采样，以研究磷酸酶活性因过氧化氢氧化而发生的变化。凭借这种独特的采样设备和无标记测定格式，带有 SAMDI 的 LCAD 提供了一种强大的新方法来监测小细胞群的细胞活动动态。

更新日期：2020-07-02

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