TY - JOUR
T1 - Development of an aptamer beacon for detection of Interferon-Gamma
AU - Tuleuova, Nazgul
AU - Jones, Caroline N.
AU - Yan, Jun
AU - Ramanculov, Erlan
AU - Yokobayashi, Yohei
AU - Revzin, Alexander
PY - 2010/3/1
Y1 - 2010/3/1
N2 - 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-γ)-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-γ (Kd = 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-γ binding with an aptamer beacon occurred within 15 min of analyte introduction-suggesting dynamic replacement of the quencher-complementary strand by IFN-γ molecules. To further highlight biosensing applications, aptamer beacon molecules were immobilized inside microfluidic channels and challenged with varying concentration of analyte. Fluorescence microscopy revealed low fluorescence in the absence of analyte and high fluorescence after introduction of IFN-γ. Importantly, unlike traditional antibody-based immunoassays, the signal was observed directly upon binding of analyte without the need for multiple washing steps, The surface immobilized aptamer beacon had a linear range from 5 to 100 nM and a lower limit of detection of 5 nM IFN-γ. In conclusion, we designed a FRET-based aptamer beacon for monitoring of an inflammatory cytokine-IFNy. In the future, this biosensing strategy will be employed to monitor dynamics of cytokine production by the immune cells.
AB - 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-γ)-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-γ (Kd = 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-γ binding with an aptamer beacon occurred within 15 min of analyte introduction-suggesting dynamic replacement of the quencher-complementary strand by IFN-γ molecules. To further highlight biosensing applications, aptamer beacon molecules were immobilized inside microfluidic channels and challenged with varying concentration of analyte. Fluorescence microscopy revealed low fluorescence in the absence of analyte and high fluorescence after introduction of IFN-γ. Importantly, unlike traditional antibody-based immunoassays, the signal was observed directly upon binding of analyte without the need for multiple washing steps, The surface immobilized aptamer beacon had a linear range from 5 to 100 nM and a lower limit of detection of 5 nM IFN-γ. In conclusion, we designed a FRET-based aptamer beacon for monitoring of an inflammatory cytokine-IFNy. In the future, this biosensing strategy will be employed to monitor dynamics of cytokine production by the immune cells.
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U2 - 10.1021/ac9025237
DO - 10.1021/ac9025237
M3 - Article
C2 - 20121141
AN - SCOPUS:77749322512
SN - 0003-2700
VL - 82
SP - 1851
EP - 1857
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 5
ER -