title: Fluorescence Detection of H5N1 Virus Gene Sequences Based on Optical Tweezers with Two-Photon Excitation Using a Single Near Infrared Nanosecond Pulse Laser.
abstract: We present an analytical platform by combining near-infrared optical tweezers with two-photon excitation for fluorescence detection of H5N1 virus gene sequences. A heterogeneous enrichment strategy, which involved polystyrene (PS) microsphere and quantum dots (QDs), was adopted. The final hybrid-conjugate microspheres were prepared by a facile one-step hybridization procedure by using PS microspheres capturing target DNA and QDs tagging, respectively. Quantitative detection was achieved by the optical tweezers setup with a low-cost 1064 nm nanosecond pulse laser for both optical trapping and two-photon excitation for the same hybrid-conjugate microsphere. The detection limits for both neuraminidase (NA) gene sequences and hemagglutinin (HA) gene sequences are 16-19 pM with good selectivity for one-base mismatch, which is approximately 1 order of magnitude lower than the most existing fluorescence-based analysis method. Besides, because of the fact that only signal from the trapped particle is detected upon two-photon excitation, this approach showed extremely low background in fluorescence detection and was successfully applied to directly detect target DNA in human whole serum without any separation steps and the corresponding results are very close to that in buffer solution, indicating the strong anti-interference ability of this method. Therefore, it can be expected to be an emerging alternative for straightforward detecting target species in complex samples with a simple procedure and high-throughput.
authors.affiliation: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, State Key Laboratory of Virology, The Institute for Advanced Studies, and Wuhan Institute of Biotechnology, Wuhan University , Wuhan, 430072, People's Republic of China.
authors.forename: Cheng-Yu
authors.initials: CY
authors.lastname: Li
authors.affiliation: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, State Key Laboratory of Virology, The Institute for Advanced Studies, and Wuhan Institute of Biotechnology, Wuhan University , Wuhan, 430072, People's Republic of China.
authors.forename: Di
authors.initials: D
authors.lastname: Cao
authors.affiliation: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, State Key Laboratory of Virology, The Institute for Advanced Studies, and Wuhan Institute of Biotechnology, Wuhan University , Wuhan, 430072, People's Republic of China.
authors.forename: Ya-Feng
authors.initials: YF
authors.lastname: Kang
authors.affiliation: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, State Key Laboratory of Virology, The Institute for Advanced Studies, and Wuhan Institute of Biotechnology, Wuhan University , Wuhan, 430072, People's Republic of China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Lin
authors.affiliation: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, State Key Laboratory of Virology, The Institute for Advanced Studies, and Wuhan Institute of Biotechnology, Wuhan University , Wuhan, 430072, People's Republic of China.
authors.forename: Ran
authors.initials: R
authors.lastname: Cui
authors.affiliation: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, State Key Laboratory of Virology, The Institute for Advanced Studies, and Wuhan Institute of Biotechnology, Wuhan University , Wuhan, 430072, People's Republic of China.
authors.forename: Dai-Wen
authors.initials: DW
authors.lastname: Pang
authors.affiliation: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, State Key Laboratory of Virology, The Institute for Advanced Studies, and Wuhan Institute of Biotechnology, Wuhan University , Wuhan, 430072, People's Republic of China.
authors.forename: Hong-Wu
authors.initials: HW
authors.lastname: Tang
mesh_terms: D001483:Base Sequence; D004279:DNA, Viral / Q000097:blood* / Q000235:genetics*; D005453:Fluorescence; D006801:Humans; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics*; D007259:Infrared Rays; D007834:Lasers; D052898:Optical Tweezers; D017785:Photons; D011137:Polystyrenes / Q000737:chemistry; D045663:Quantum Dots; D013997:Time Factors
chemical_list: D004279:DNA, Viral; D011137:Polystyrenes