Selection of aptamers based on a protein microarray integrated with a microfluidic chip.

Abstract

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_d). 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^-1 and the detection limit was 0.39 μg mL^-1. Thus, the innovative PMM-SELEX presented shows stability, accuracy and high efficiency for aptamer screening.