Browsing by Author "Sun, Yuh-Chang"

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Gold nanoparticle-based inductively coupled plasma mass spectrometry amplification and magnetic separation for the sensitive detection of a virus-specific RNA sequence
(2011-04-08)
Hsu, I-Hsiang
;
Chen, Wen-Hung
;
Wu, Tung-Kung
;
Sun, Yuh-Chang
;
生物科技學系
We have developed a sensitive gold nanoparticle (AuNP)-based inductively coupled plasma mass spectrometry (ICP-MS) amplification and magnetic separation method for the detection of oligonucleotide sequences. The assay relies on (i) the sandwich-type binding of two designed probe sequences that specifically recognize the target oligonucleotide sequences, (ii) magnetic bead separation, and (iii) AuNP-based ICP-MS amplification detection. To enhance the analytical signal and minimize the background signal resulting from nonspecific binding, we performed a series of experiments to evaluate the effects of various parameters (the concentration of the capture probe; the time required for hybridization; the number of washings required to eliminate nonspecific binding) on the oligonucleotide detection. Under the optimized conditions, the detection limit was 80 zmol (corresponding to 1.6fM of the target sequence in a sample volume of 50 mu L). Moreover, it employs a shorter hybridization step and ICP-MS, this procedure is relatively simple and rapid (ca. 1.5 h). Based on the analytical results obtained using complementary and mismatched sequences, our method exhibits good performance in distinguishing complementary and random oligonucleotides. Compared with the "gold standard" methodology (plaque assay) for the quantification of dengue virus, our method has the capability to allow early detection of dengue virus in complicated and small-volume samples, with high specificity, good analytical sensitivity, and superior time-effectiveness. (C) 2011 Elsevier B.V. All rights reserved.
Immunocapture couples with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for rapid detection of type 1 dengue virus
(2013-05-03)
Chen, Wen-Hung
;
Hsu, I-Hsiang
;
Sun, Yuh-Chang
;
Wang, Yu-Kuo
;
Wu, Tung-Kung
A facile method for accurate detection of type I dengue virus (DV1) infection from complex biological mixtures, using type specific immunocapture coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), was developed. The biological mixtures were treated with magnetic particles coated with a monoclonal antibody directly against type 1 dengue virus. After immunocapture purification, the DV1 was eluted with 30% acetic acid, directly spotted with seed-layer method, and analyzed by MALDI-TOF MS for DV1 capsid protein. The detection limit of the assay was similar to 10(5) pfu/mL by MALDI-TOF MS. The immunocapture could unambiguously differentiate the DV1 from other serotypes of the dengue viruses and Japanese encephalitis virus, and could be used as a specific probe to detect DV1 from complex biological mixtures. (C) 2013 Elsevier B.V. All rights reserved.