Development of an aptamer beacon for detection of interferon-gamma

Analytical Chemistry
Nazgul TuleuovaAlexander Revzin

Abstract

Traditional antibody-based affinity sensing strategies employ multiple reagents and washing steps and are unsuitable for real-time detection of analyte binding. Aptamers, on the other hand, may be designed to monitor binding events directly, in real-time, without the need for secondary labels. The goal of the present study was to design an aptamer beacon for fluorescence resonance energy transfer (FRET)-based detection of interferon-gamma (IFN-gamma)--an important inflammatory cytokine. Variants of DNA aptamer modified with biotin moieties and spacers were immobilized on avidin-coated surfaces and characterized by surface plasmon resonance (SPR). The SPR studies showed that immobilization of aptamer via the 3' end resulted in the best binding IFN-gamma (K(d) = 3.44 nM). This optimal aptamer variant was then used to construct a beacon by hybridizing fluorophore-labeled aptamer with an antisense oligonucleotide strand carrying a quencher. SPR studies revealed that IFN-gamma binding with an aptamer beacon occurred within 15 min of analyte introduction--suggesting dynamic replacement of the quencher-complementary strand by IFN-gamma molecules. To further highlight biosensing applications, aptamer beacon molecules were immobilized i...Continue Reading

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Citations

Jun 23, 2012·Analytical and Bioanalytical Chemistry·Ying LiuAleksandr L Simonian
Sep 8, 2010·Analytical Chemistry·Ying LiuAlexander Revzin
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Dec 21, 2010·Cellular and Molecular Bioengineering·Nazgul Tuleuova, Alexander Revzin
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