title: 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 (Kd). 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-1and the detection limit was 0.39 μg mL-1. Thus, the innovative PMM-SELEX presented shows stability, accuracy and high efficiency for aptamer screening.
authors.affiliation: State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, China. lihui@nju.edu.cn xudanke@nju.edu.cn.
authors.forename: Xiaohui
authors.initials: X
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, China. lihui@nju.edu.cn xudanke@nju.edu.cn.
authors.forename: Hui
authors.initials: H
authors.lastname: Li
authors.affiliation: State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, China. lihui@nju.edu.cn xudanke@nju.edu.cn.
authors.forename: Wenchao
authors.initials: W
authors.lastname: Jia
authors.affiliation: State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, China. lihui@nju.edu.cn xudanke@nju.edu.cn.
authors.forename: Zhu
authors.initials: Z
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, China. lihui@nju.edu.cn xudanke@nju.edu.cn.
authors.forename: Danke
authors.initials: D
authors.lastname: Xu
mesh_terms: D000818:Animals; D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000378:metabolism; D002417:Cattle; D004277:DNA, Single-Stranded / Q000378:metabolism; D005454:Fluorescence Polarization; D007700:Kinetics; D056656:Lab-On-A-Chip Devices; D007781:Lactoferrin / Q000032:analysis; D008892:Milk / Q000378:metabolism; D016133:Polymerase Chain Reaction; D040081:Protein Array Analysis / Q000295:instrumentation / Q000379:methods*; D052156:SELEX Aptamer Technique; D020349:Surface Plasmon Resonance
chemical_list: D052157:Aptamers, Nucleotide; D004277:DNA, Single-Stranded; D007781:Lactoferrin