We developed an efficient and fast method based on a protein microarray integrated with a microfluidic chip for the process of SELEX (systematic evolution of ligands by exponential enrichment). Lactoferrin from bovine milk was used as a target protein, while bovine serum albumin (BSA), α-lactalbumin, β-lactoglobulin and casein were used as negative proteins. They were separately dotted and immobilized to prepare the protein microarray and the resulting microarray was further integrated into a microfluidic chip for the SELEX (PMM-SELEX) process. The interaction between aptamer candidates and targets could be monitored using a fluorescence microarray scanner and the whole PMM-SELEX process was performed through seven-round selection. As a result, five aptamers (Lac-14, Lac-6a, Lac-9, Lac-5, Lac-3a) with high specificity and affinity can be repeatedly obtained during three times of independent repeated selection. Surface plasmon resonance (SPR) was used to calculate the dissociation constants (K). The aptamer Lac-6a was then used for detection of lactoferrin by fluorescence polarization. A linear response was observed for lactoferrin concentrations in the range of 0.78-50 μg mL and the detection limit was 0.39 μg mL. Thus, the innovative PMM-SELEX presented shows stability, accuracy and high efficiency for aptamer screening.

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 (AgNPs). The split aptamers could bind to different sites of Lac and assemble into a split-aptamers-target complex, narrowing the distance between AgNPs and FITC dye. As a result, AgNPs could produce a mass-augmented and metal-enhanced fluorescence (MEF) effect. In general, ternary amplification based on AgNPs, 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.

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