DNA aptamer-based bioanalysis of IgE by fluorescence anisotropy.
A rapid, homogeneous aptamer-based bioanalysis is reported for the sensitive detection of immunoglobulin E (IgE) using fluorescence polarization (FP). 5'-End-labeled D17.4 DNA aptamer was used for IgE detection based on the anisotropy differences of the labeled ligand. Two different fluorophores, fluorescein and Texas Red, were used to analyze IgE in the low-nanomolar range with high specificity. Measurable anisotropy changes were observed with a short equilibration time. Analysis of the binding data reveals a possible cooperative binding process in solution. The nature of the fluorophore clearly influences the sensitivity of the analysis more than the tether length used for the dye conjugation. The local fluorophore motion is seen to influence the sensitivity of the FP probe significantly. Texas Red is seen to be relatively more sensitive for this approach and has apparently favorable dye-DNA interactions, and a limit of detection of 350 pM was obtained. Significant temperature dependence of the FP responses has been observed in this work. Ionic composition of the binding buffer also influences the assay sensitivity. The results confirm the promise and potential of similar homogeneous assays for aptamer-based bioanalysis.
Gokulrangan
Giridharan
G
Department of Chemistry, University of Kansas, Lawrence, Kansas 66045, USA.
Unruh
Jay R
JR
Holub
Douglas F
DF
Ingram
Brian
B
Johnson
Carey K
CK
Wilson
George S
GS
D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000378:metabolism*; D019793:Fluorescein / Q000737:chemistry; D005454:Fluorescence Polarization / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D007073:Immunoglobulin E / Q000032:analysis* / Q000378:metabolism; D012680:Sensitivity and Specificity; D013696:Temperature; D014966:Xanthenes / Q000737:chemistry
D052157:Aptamers, Nucleotide; D005456:Fluorescent Dyes; D014966:Xanthenes; D007073:Immunoglobulin E; C034657:Texas red; D019793:Fluorescein