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

• Published in: Talanta (Oxford) Vol. 193; pp. 110 - 117
• Main Authors: Yu, Fang, Li, Hui, Sun, Wei, Zhao, Yaju, Xu, Danke, He, Fuchu
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-02-2019
• Subjects: Aptamer; Fluorescence activated cell sorting; Lactoferrin; SELEX; Silver decahedral nanoparticles; Silver decahedral nanoparticles; Lactoferrin; Aptamer; Fluorescence activated cell sorting; SELEX
• ISSN: 0039-9140; 1873-3573
• DOI: 10.1016/j.talanta.2018.09.063

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 PSLac, PSα-Lac, PSβ-Lac, PSBSA and PSCas. PSLac was firstly interacted with Cy5 labeled library (Lib), then hybridized with Cy5 modified silver decahedral nanoparticles (AgNPCy5) to form PSLac/Lib/AgNPCy5 conjugates. FACS was used to separate and collect PSLac/Lib/AgNPCy5 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 (Kd) 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.
We developed a novel method for efficiently screening aptamers targeted lactoferrin from a complex ssDNA library based on silver decahedral nanoparticles (AgNP) and fluorescence activated cell sorting (FACS). Six aptamers (Ylac1, Ylac4, Ylac5, Ylac6, Ylac8 and Ylac9) were obtained after five-round of selection, which showed good specificity towards lactoferrin. And the equilibrium dissociation constants (Kd) of six aptamers were calculated and all were in the nanomolar range. [Display omitted]
•AgFACS-SELEX is based on fluorescence activated cell sorting and silver nanoparticle.•It improved in situ monitoring ability of high sensitivity and screening specificity.•Kd of the selected six aptamers were all in the nanomolar range.