Convenient Construction of Orthogonal Dual Aptamer-Based Plasmonic Immunosandwich Assay for Probing Protein Disease Markers in Complex Samples and Living Animals.

Aptamers, because of their outstanding merits including simple synthesis and easy modification, have been widely used as antibody alternatives to construct novel immunosandwich assays. Dual aptamer-based sandwich assays exhibit multiple advantages over conventional immunosandwich assays and single aptamer-based sandwich assays. However, their construction is hampered by the limited knowledge of binding orthogonality of aptamers reported in the literature. Herein, we present a new strategy for conveniently constructing an orthogonal dual aptamer-based plasmonic immunosandwich assay (odA-PISA) for probing proteins in complex samples and living animals. An orthogonal aptamer pair was first efficiently selected from the aptamers reported in the literature by affinity capillary electrophoresis. Then, a target protein-capturing gold thin-layer-coated probe and silver nanoparticle-based Raman labeling nanotags were conveniently prepared with the selected aptamers and used to construct the assay. The double aptamers used ensured the specificity, whereas the plasmonic coupling effect between the target-capturing probe and the generated Raman nanotags significantly enhanced the Raman signal intensity, providing high sensitivity. As a proof of principle, alkaline phosphatase (ALP) was used as the target. The constructed odA-PISA exhibited high specificity and high sensitivity toward ALP, giving cross-reactivity ≤ 4.2% and the limit of detection of 3.8 pM (S/N = 4). The quantitative determination of ALP in human serum and probing ALP in tumor-bearing mice were achieved, showing the great application potential of the method. This strategy is widely applicable to other protein disease markers. Therefore, it opened a new access to the construction of sensitive dual aptamer-based sandwich assays for real-world applications, particularly disease diagnosis.