title: Sensitive multiplexed DNA detection using silica nanoparticles as the target capturing platform.
abstract: We present a simple and sensitive method for multiplexed DNA detection by simultaneously capturing two different DNA sequences with a same silica nanoparticle (NP) through a sandwich mode. This biobarcode assay method was demonstrated by using oligonucleotide sequences of 64 bases associated with human papillomavirus (HPV) 16 and 18 L1 genes as model systems. The nonfluorescent carboxyl-modified silica NPs were prepared using water-in-oil (W/O) microemulsion methods. Avidin was immobilized on the surface of the NPs by covalent binding to the carboxyl linkers. The binding capacity of the avidin-covered NPs for ligand biotin was quantified and the results show that about 8 avidin molecules are bound to one nanoparticle. The silica nano-platforms were prepared through the biotin-avidin interaction and the amounts of capture DNA strands for HPV-16 and HPV-18 (C-16 and C-18, respectively) conjugated to the surface of the same NPs were measured using fluorescent dye hoechst33258. The calculated result shows that the amounts of conjugated C-16 and C-18 on 1mg of NPs (9.2 pmol) are about 13.5 pmol and 15.5 pmol, respectively. A one-step hybridization reaction was performed by mixing the silica nano-platforms, HPV-16 and HPV-18 target DNA (T-16 and T-18), fluorescein amidite (FAM) or 6-carboxyl-X-rhodamine (Rox) labeled HPV-16 and HPV-18 probes. The hybrid-conjugated NPs were separated by centrifugation, and T-16 and T-18 were detected by measuring fluorescence signals of FAM and Rox respectively. The results show linear dependence of the fluorescence intensity on target DNA concentration in the range from 0.5 to 9 nM, and the detection limit (3σ) of T-16 and T-18 is 0.17 nM and 0.78 nM, respectively.
authors: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, State Key Laboratory of Virology, and Wuhan Institute of Biotechnology, Wuhan University, Wuhan 430072, PR China.
authors: Wei
authors: W
authors: Wang
authors: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, State Key Laboratory of Virology, and Wuhan Institute of Biotechnology, Wuhan University, Wuhan 430072, PR China.
authors: Dai-Wen
authors: DW
authors: Pang
authors: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, State Key Laboratory of Virology, and Wuhan Institute of Biotechnology, Wuhan University, Wuhan 430072, PR China. Electronic address: hwtang@whu.edu.cn.
authors: Hong-Wu
authors: HW
authors: Tang
mesh_terms: D001360:Avidin / Q000737:chemistry; D001710:Biotin / Q000737:chemistry; D004247:DNA / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D015342:DNA Probes / Q000737:chemistry / Q000235:genetics; D005452:Fluoresceins / Q000737:chemistry; D052162:Human papillomavirus 16 / Q000235:genetics; D052161:Human papillomavirus 18 / Q000235:genetics; D053768:Metal Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission; D015203:Reproducibility of Results; D012822:Silicon Dioxide / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D015342:DNA Probes; D005452:Fluoresceins; D001360:Avidin; D001710:Biotin; D012822:Silicon Dioxide; D004247:DNA
keywords: Hoechst33258; Human papillomavirus; Multiplexed DNA detection; Nano-platform; Silica nanoparticles