Selection of aptamers against Lactoferrin based on silver enhanced and fluorescence-activated cell sorting

Abstract

We report a novel method for efficiently screening aptamers from a complex ssDNA library based on silver decahedral nanoparticles (AgNP) and fluorescence activated cell sorting (FACS). In this method, target protein (lactoferrin) and negative proteins (α-lactalbumin, β-lactoglobulin, bovine serum albumin, casein) were respectively immobilized on polystyrene microspheres (PS) to form PS^Lac, PS^α-Lac, PS^β-Lac, PS^BSA and PS^Cas. PS^Lac was firstly interacted with Cy5 labeled library (Lib), then hybridized with Cy5 modified silver decahedral nanoparticles (AgNP^Cy5) to form PS^Lac/Lib/AgNP^Cy5 conjugates. FACS was used to separate and collect PS^Lac/Lib/AgNP^Cy5 conjugates from complicated complex. AgNP was used to increase the fluorescence intensity in the selecting process and choose non-self-hybridization of Lib. Six aptamers (Ylac1, Ylac4, Ylac5, Ylac6, Ylac8 and Ylac9) were obtained after five-round of selection. These aptamers showed good specificity towards lactoferrin in the presence of negative proteins. The equilibrium dissociation constants (K_d) of six aptamers were calculated and all were in the nanomolar range. In a word, AgNP-FACS SELEX (AgFACS-SELEX) is a rapid, sensitive and highly efficient method for screening aptamers.

Keywords: Aptamer; Fluorescence activated cell sorting; Lactoferrin; SELEX; Silver decahedral nanoparticles.