Targeted permeabilization of the cell wall and extraction of charged molecules from single cells in intact plant clusters using a focused electric field,Analyst

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Targeted permeabilization of the cell wall and extraction of charged molecules from single cells in intact plant clusters using a focused electric field
Analyst ( IF 3.6 ) Pub Date : 2021-2-11 , DOI: 10.1039/d0an02163f
Sangamithirai Subramanian Parimalam _{1,

2,

3,

4,

5} , Mahmoud Nady Abdelmoez _{1,

2,

3} , Arata Tsuchida _{1,

2,

3,

4,

5} , Naoyuki Sotta _{3,

6,

7,

8} , Mayuki Tanaka _{3,

6,

7,

8} , Takashi Kuromori _{3,

9,

10} , Toru Fujiwara _{3,

6,

7,

8} , Masami Yokota Hirai _{3,

10,

11} , Ryuji Yokokawa _{3,

4,

5,

12} , Yusuke Oguchi _{1,

2,

3} , Hirofumi Shintaku _{1,

2,

3}

Affiliation

The extraction of cellular contents from plant cells covered with cell walls remains a challenge, as it is physically hindered by the cell wall. We present a new microfluidic approach that leverages an intense pulsed electric field for permeabilizing the cell wall and a focused DC electric field for extracting the cellular contents selectively from a few targeted cells in a cluster of intact plant cells. We coupled the approach with on-chip fluorescence quantification of extracted molecules leveraging isotachophoresis as well as off-chip reverse transcription-quantitative polymerase chain reaction detecting extracted mRNA molecules. Our approach offers a workflow of about 5 min, isolating a cluster of intact plant cells, permeabilizing the cell wall, selectively extracting cytosolic molecules from a few targeted cells in the cluster, and outputting them to off-chip analyses without any enzymatic reactions. We anticipate that this approach will create a new opportunity to explore plant biology through less biased data realized by the rapid extraction of molecules from intact plant clusters.

中文翻译：

使用聚焦电场对细胞壁进行有针对性的透化，并从完整植物簇中的单个细胞中提取带电分子

从被细胞壁覆盖的植物细胞中提取细胞内容物仍然是一个挑战，因为它在物理上受到细胞壁的阻碍。我们提出了一种新的微流控方法，该方法利用强脉冲电场渗透细胞壁，并利用聚焦直流电场选择性地从完整植物细胞簇中的一些靶细胞中提取细胞内容物。我们将这种方法与利用等速电泳的提取分子的芯片上荧光定量以及芯片外逆转录定量聚合酶链反应检测提取的mRNA分子相结合。我们的方法提供了大约5分钟的工作流程，隔离了完整的植物细胞簇，透化了细胞壁，从簇中的一些目标细胞中选择性地提取了胞质分子，并将其输出到芯片外分析，而没有任何酶促反应。我们预计，这种方法将为通过从完整植物簇中快速提取分子而实现的较少偏倚数据创造一个探索植物生物学的新机会。

更新日期：2021-02-24

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