Microfluidics and Nanofluidics

, Volume 19, Issue 4, pp 795–804 | Cite as

Isolation of thermally sensitive protein-binding oligonucleotides on a microchip

  • John P. Hilton
  • Timothy Olsen
  • Jinho Kim
  • Jing Zhu
  • ThaiHuu Nguyen
  • Mihaela Barbu
  • Renjun Pei
  • Milan Stojanovic
  • Qiao Lin
Research Paper

Abstract

This paper presents a microfluidic chip capable of isolating thermally sensitive protein-binding aptamer candidates. The chip makes use of bead-immobilized target molecules and DNA (deoxyribonucleic acid) sequences to enable a simplified chip design, in which affinity selection and PCR (polymerase chain reaction) amplification of target-binding sequences occur in temperature-controlled microchambers. Using pressure-driven flow, buffer containing single-stranded DNA molecules with randomized sequences is cycled through a series of affinity selection and PCR amplification steps on microbeads. Successive introduction of the sample to each chamber effects a process of competition whereby DNA strands with weak binding strength to target molecules are rejected in favor of strongly binding sequences. Using bead-based PCR, the amplification step was miniaturized and integrated with affinity selection, resulting in significant reductions in process time and reagent use. As a demonstration, temperature-dependent selection and amplification of single-stranded oligonucleotides that bind to human Immunoglobulin E (IgE) was performed in 4 h, a 20-fold reduction in process time as compared to conventional methods that would require approximately a week. Fluorescent binding assays then demonstrated that the desired temperature specificity was imparted to the aptamer candidates within just one round of selection, and within two rounds the aptamer candidates exhibited enhanced affinity toward IgE.

Keywords

Aptamers Microfluidics Proteins SELEX Temperature dependence 

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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • John P. Hilton
    • 1
  • Timothy Olsen
    • 1
  • Jinho Kim
    • 1
  • Jing Zhu
    • 1
  • ThaiHuu Nguyen
    • 1
  • Mihaela Barbu
    • 2
  • Renjun Pei
    • 3
  • Milan Stojanovic
    • 2
  • Qiao Lin
    • 1
  1. 1.Department of Mechanical EngineeringColumbia UniversityNew YorkUSA
  2. 2.Division of Clinical Pharmacology and Experimental Therapeutics, Department of MedicineColumbia UniversityNew YorkUSA
  3. 3.Suzhou Institute of Nano-Tech and Nano-BionicsChinese Academy of SciencesSuzhouChina

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