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Probing cytoplasmic and nuclear microRNAs in single living cells via plasmonic affinity sandwich assay

Jia Liu1, Yanrong Wen1, Hui He1

  • 1State Key Laboratory of Analytical Chemistry for Life Science , School of Chemistry and Chemical Engineering , Nanjing University , 163 Xianlin Avenue , Nanjing 210023 , China .

Chemical Science
|October 6, 2018
PubMed

Insights

This study introduces a novel plasmonic affinity sandwich assay (PASA) for detecting microRNAs (miRNAs) within single living cells. PASA enables rapid, ultrasensitive, and subcellularly resolved analysis of cytoplasmic and nuclear miRNAs.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biotechnology

Background:

  • MicroRNAs (miRNAs) are crucial regulators of gene expression, and understanding their subcellular localization is vital for cell biology.
  • Existing methods for analyzing subcellular miRNAs in living cells lack resolution, compromise cell viability, or alter cellular composition.
  • Subcellular miRNA distribution contributes to cellular heterogeneity, necessitating advanced analytical techniques.

Purpose of the Study:

  • To develop a rapid and ultrasensitive method for probing subcellular microRNAs in single living cells.
  • To achieve subcellular resolution in miRNA detection without compromising cell integrity.
  • To enable simultaneous analysis of cytoplasmic and nuclear miRNAs within intact cells.

Main Methods:

  • Development of a plasmonic affinity sandwich assay (PASA) combining in vivo subcellular extraction and plasmon-enhanced Raman scattering (PERS) detection.
  • Utilizing a micromanipulator and gold-coated microprobes for specific extraction of target miRNAs from cytoplasm and nucleus.
  • Employing silver nanotags with Raman reporters for ultrasensitive detection of miRNA-probe complexes.

Main Results:

  • PASA demonstrated high subcellular resolution, differentiating nuclear miR-29b from cytoplasmic miR-29a.
  • The method provided quantitative capabilities for cytoplasmic miRNAs (miR-21, miR-155, miR-203).
  • Analysis required only 10 minutes, bypassing traditional subcellular fractionation and amplification steps.

Conclusions:

  • PASA is a rapid, ultrasensitive, and high-resolution technique for analyzing subcellular miRNAs in single living cells.
  • This method offers a promising tool for investigating miRNA functions, localizations, and cellular heterogeneity.
  • PASA overcomes limitations of previous methods, preserving cell viability and composition during analysis.

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