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An automatic microfluidic system for rapid screening of cancer stem-like cell-specific aptamers

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Abstract

This study reports a microfluidic system which automatically performs the systematic evolution of ligands by exponential enrichment (SELEX) process for rapid screening of aptamers which are specific to cancer stem-like cells. The system utilizes magnetic bead-based techniques to select DNA aptamers and has several advantages including a rapid, automated screening process, and less consumption of cells and reagents. By integrating a microfluidic control module, a magnetic bead-based aptamer extraction module, and a temperature control module, the entire Cell-SELEX process can be performed in a shorter period of time. Compared with the traditional Cell-SELEX process, this microfluidic system is more efficient and consumes fewer sample volumes. It only takes approximately 3 days for an entire Cell-SELEX process with 15 screening runs, which is relatively faster than that of a traditional Cell-SELEX process (1 week for 15 rounds). The binding affinity of this resulting specific aptamer was measured by a flow cytometric analysis to have a dissociation constant (K d) of 15.32 nM. The capture rate for cancer stem-like cells using the specific aptamer-conjugated bead is better than that using Ber-EP4 antibody-conjugated bead. This microfluidic system may provide a powerful platform for the rapid screening of cell-specific aptamers.

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Abbreviations

A549shEcad :

Human lung cancer cells (A549) treated with E-cadherin specific shRNA (shEcad) as target cells

A549shLuc :

A549 cell treated with shRNA targeting Luciferase (shLuc) as control cells

ATCC:

American type culture collection

Ber-EP4:

Mouse anti-human EpCAM mAbs

bp:

Base-pairs

BSA:

Bovine serum albumin

CNC:

Computer-numerical-control

dNTPs:

Deoxynucleotide triphosphates

DPBS:

Dulbecco’s phosphate-buffered saline

EMVs:

Electromagnetic valves

EpCAM:

Epithelial cell adhesion molecule

FBS:

Fetal bovine serum

FITC:

Fluorescein isothiocyanate

K d :

Dissociation constant

mAbs:

Monoclonal antibodies

PCR:

Polymerase chain reaction

PDMS:

Polydimethylsiloxane

RNA:

Ribonucleic acid

SELEX:

Systematic evolution of ligands by exponential enrichment

SSCT buffer:

750 mM NaCl, 75 mM sodium citrate, 0.05 % Tween-20, pH 7.0

ssDNA:

Single-stranded deoxyribonucleic acid

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Acknowledgments

The authors would like to thank the National Science Council in Taiwan (NSC 100-2120-M-007-014) and the “Toward A World-class University” Project for financial support of this study.

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Authors and Affiliations

  1. Department of Microbiology and Immunology, National Cheng Kung University, Tainan, 70101, Taiwan

    Chen-Hsun Weng

  2. Institute of Basic Medical Sciences, National Cheng Kung University, Tainan, 70101, Taiwan

    I-Shan Hsieh

  3. Institute of Oral Medicine, National Cheng Kung University, Tainan, 70101, Taiwan

    Yuh-Ling Chen

  4. Department of Medical Laboratory Science and Biotechnology, National Cheng Kung University, Tainan, 70101, Taiwan

    Hsin-I Lin & Shu-Chu Shiesh

  5. Department of Power Mechanical Engineering, National Tsing Hua University, Hsinchu, 30013, Taiwan

    Lien-Yu Hung & Gwo-Bin Lee

Authors
  1. Chen-Hsun Weng
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  2. I-Shan Hsieh
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  3. Lien-Yu Hung
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  4. Hsin-I Lin
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  5. Shu-Chu Shiesh
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  6. Yuh-Ling Chen
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  7. Gwo-Bin Lee
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Corresponding authors

Correspondence to Yuh-Ling Chen or Gwo-Bin Lee.

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Weng, CH., Hsieh, IS., Hung, LY. et al. An automatic microfluidic system for rapid screening of cancer stem-like cell-specific aptamers. Microfluid Nanofluid 14, 753–765 (2013). https://doi.org/10.1007/s10404-012-1095-3

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  • DOI: https://doi.org/10.1007/s10404-012-1095-3

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