Isolation of thermally sensitive protein-binding oligonucleotides on a microchip

John P. Hilton, Timothy Olsen, Jinho Kim, Jing Zhu, Thai Huu Nguyen, Mihaela Barbu, Renjun Pei, Milan Stojanovic, Qiao Lin

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

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.

Original languageEnglish
Pages (from-to)795-804
Number of pages10
JournalMicrofluidics and Nanofluidics
Volume19
Issue number4
DOIs
StatePublished - 1 Oct 2015

Keywords

  • Aptamers
  • Microfluidics
  • Proteins
  • SELEX
  • Temperature dependence

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