Aptamer sandwich assays: human α-thrombin detection using liposome enhancement

Fluorescent dye-encapsulating liposomes tagged with aptamers were developed and used as reporting signals in an aptamer-based sandwich assay. alpha-Thrombin was utilized as a prototypical analyte as two well-studied aptamers binding distinct epitopes are available to form a sandwich complex. Cholesteryl-TEG-modified aptamers were embedded into the liposomal lipid bilayer while the interior cavity of the liposomes encapsulated fluorescent sulforhodamine B dye. Such liposomes successfully formed a sandwich complex with alpha-thrombin and a microtiter plate immobilized aptamer, proving that aptamers retain their ability to fold when anchored to the liposome surface. Parameters studied included liposomal aptamer coverage, sandwich aptamer orientation, aptamer label orientation, aptamer spacer length and type, incubation buffer, and aptamer concentration. The optimized conditions found here in the fluorescence assay led to a limit of detection of 64 pM or 2.35 ng/mL, corresponding to 6.4 fmol or 235 pg, respectively, in a 100 mu L volume. This is an order of magnitude lower than previous sandwich aptamer assays using the same sequences with lowest reported limits of detection of 0.45 nM. In addition, the assay was applied successfully to the detection of alpha-thrombin in human plasma. The success of this method in a standard microtiter plate format and the relatively facile functionalization of liposomes with aptamers suggest that this approach provides a versatile option for routine analytical applications.