title: A cytometric assay for ultrasensitive and robust detection of human telomerase RNA based on toehold strand displacement.
abstract: Human telomerase RNA (hTR), works as a template for synthesis of telomeric DNA repeats at the ends of linear eukaryotic chromosomes, is overexpressed in tumor cells and its concentration has a positive correlation with telomerase activity. The lack of facile and reliable method for detection of hTR in complex matric limited its application for clinical diagnosis. To address the limitation, herein, we proposed a facile and reliable flow cytometric assay for sensitive and specific detection of hTR by combing magnetic enrichment with signal amplification of DNA toehold strand displacement reaction (TSDR). Two hairpin DNA probes of TSDR are ingeniously designed, including biotinylated hairpin DNA1 (H1) and carboxyfluorescein (FAM)-labeled hairpin DNA2 (F-H2). Firstly, H1 was immobilized on streptavidin-functionalized magnetic beads (STV-MBs) through biotin-avidin interaction. In the presence of hTR DNA, TSDR between H1 and F-H2 was triggered to continuously form H1/H2 duplex, resulting in a "turn on" fluorescence on the surface of MBs. Due to fluorescence amplification of TSDR and magnetic enrichment, hTR-DNA can be sensitively, specifically and facile analyzed by flow cytometry and fluorescence microscopy imaging. The detection limit of flow cytometry is 0.3pM, which is superior to those of most existing approaches. Moreover, the proposed strategy can be successfully utilized to detect hTR in complex biological media as well. Therefore, an enzyme-free amplification approach is provided for robust and rapid detecting hTR DNA, which offers a facile, reliable and sensitive method for studying disease-related gene.
authors.affiliation: Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an, 710062 China.
authors.forename: Jing
authors.initials: J
authors.lastname: Xu
authors.affiliation: Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an, 710062 China.
authors.forename: Yanjun
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an, 710062 China.
authors.forename: Luzhu
authors.initials: L
authors.lastname: Yang
authors.affiliation: Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an, 710062 China.
authors.forename: Yanfang
authors.initials: Y
authors.lastname: Gao
authors.affiliation: Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an, 710062 China.
authors.forename: Baoxin
authors.initials: B
authors.lastname: Li
authors.affiliation: Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an, 710062 China. Electronic address: jinyan@snnu.edu.cn.
authors.forename: Yan
authors.initials: Y
authors.lastname: Jin
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D057230:Limit of Detection; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D061848:Optical Imaging / Q000379:methods; D012313:RNA / Q000032:analysis*; D019098:Telomerase / Q000032:analysis*
chemical_list: C422952:telomerase RNA; D012313:RNA; D019098:Telomerase
keywords: Flow cytometry; Fluorescence imaging; Telomerase RNA; Toehold strand displacement reaction