Bivalent Aptasensor Based on Silver-Enhanced Fluorescence Polarization for Rapid Detection of Lactoferrin in Milk

Here we report a novel type of bivalent aptasensor based on silver-enhanced fluorescence polarization (FP) for detection of lactoferrin (Lac) in milk powder with high sensitivity and specificity. The novel two split aptamers were obtained from the aptamer reported in our previous SELEX (systematic evolution of ligands by exponential enrichment) selection, and their minimal structural units were optimized on the basis of their affinity and specificity. Also, dual binding sites of split aptamers were verified. The bivalent aptamers were modified to be linked with signal-molecule fluorescein isothiocyanate (FITC) and enhancer silver decahedral nanoparticles (Ag₁₀NPs). The split aptamers could bind to different sites of Lac and assemble into a split-aptamers-target complex, narrowing the distance between Ag₁₀NPs and FITC dye. As a result, Ag₁₀NPs could produce a mass-augmented and metal-enhanced fluorescence (MEF) effect. In general, ternary amplification based on Ag₁₀NPs, split aptamers, and the MEF effect all contributed to the significant increase of FP values. It was proved that the sensitivity of this assay was about 3 orders of magnitude over traditional aptamer-based homogeneous assays with a detection limit of 1.25 pM. Furthermore, this design was examined by actual milk powder with rapid and high-throughout detection.