Browsing by Author "Chen, Yu-Shiun"

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A protein transistor made of an antibody molecule and two gold nanoparticles
(2012-03-01)
Chen, Yu-Shiun
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Hong, Meng-Yen
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Huang, G. Steven
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材料科學與工程學系
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生醫電子轉譯研究中心
A major challenge in molecular electronics is to attach electrodes to single molecules in a reproducible manner to make molecular junctions that can be operated as transistors. Several attempts have been made to attach electrodes to proteins, but these devices have been unstable. Here, we show that self-assembly can be used to fabricate, in a highly reproducible manner, molecular junctions in which an antibody molecule (immunoglobulin G) binds to two gold nanoparticles, which in turn are connected to source and drain electrodes. We also demonstrate effective gating of the devices with an applied voltage, and show that the charge transport characteristics of these protein transistors are caused by conformational changes in the antibody. Moreover, by attaching CdSe quantum dots to the antibody, we show that the protein transistor can also be gated by an applied optical field. This approach offers a versatile platform for investigations of single-molecule-based biological functions and might also lead to the large-scale manufacture of integrated bioelectronic circuits.
A protein transistor made of an antibody molecule and two gold nanoparticles (vol 7, pg 197, 2012)
(2012-04-01)
Chen, Yu-Shiun
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Hong, Meng-Yen
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Huang, G. Steven
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生醫電子轉譯研究中心
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Biomedical Electronics Translational Research Center
Application of Nanobiotechnology to Construct a Piezoelectric Sensor Matrix Sensing the Flexibility of Immunoglobulin
(2007)
Huang, G. Steven
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Chen, Yu-Shiun
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Lin, Xin-Yau
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材料科學與工程學系奈米科技碩博班
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Graduate Program of Nanotechnology , Department of Materials Science and Engineering
We have constructed a piezoelectric sensor matrix with the capability of measuring the flexibility of immunoglobulin. The matrix consisted of 6 independent quartz crystal microbalance (QCM) coated with gold nanoparticles (GNP) of 3.5, 5, 12, 17, and 37 nm, and BSA. The detection was performed simultaneously. Antiserum against GNP bound to QCMs coated with 3.5 nm GNP and maximized for 5 nm GNP-coated QCM. To our best knowledge, this is the first biosensor sensing the topological change and flexibility of biological macromolecule.
Assessment of gold nanoparticles as a size-dependent vaccine carrier for enhancing the antibody response against synthetic foot-and-mouth disease virus peptide
(2010-05-14)
Chen, Yu-Shiun
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Hung, Yao-Ching
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Lin, Wei-Hsu
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Huang, Guewha Steven
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材料科學與工程學系
To assess the ability of gold nanoparticles (GNPs) to act as a size-dependent carrier, a synthetic peptide resembling foot-and-mouth disease virus (FMDV) protein was conjugated to GNPs ranging from 2 to 50 nm in diameter (2, 5, 8, 12, 17, 37, and 50 nm). An extra cysteine was added to the C-terminus of the FMDV peptide (pFMDV) to ensure maximal conjugation to the GNPs, which have a high affinity for sulfhydryl groups. The resultant pFMDV-GNP conjugates were then injected into BALB/c mice. Immunization with pFMDV-keyhole limpet hemocyanin (pFMDV-KLH) conjugate was also performed as a control. Blood was obtained from the mice after 4, 6, 8, and 10 weeks and antibody titers against both pFMDV and the carriers were measured. For the pFMDV-GNP immunization, specific antibodies against the synthetic peptide were detected in the sera of mice injected with 2, 5, 8, 12, and 17 nm pFMDV-GNP conjugates. Maximal antibody binding was noted for GNPs of diameter 8-17 nm. The pFMDV-GNPs induced a three-fold increase in the antibody response compared to the response to pFMDV-KLH. However, sera from either immunized mouse group did not exhibit an antibody response to GNPs, while the sera from pFMDV-KLH-immunized mice presented high levels of binding activity against KLH. Additionally, the uptake of pFMDV-GNP in the spleen was examined by inductively coupled plasma mass spectroscopy (ICP-MS) and transmission electron microscopy (TEM). The quantity of GNPs that accumulated in the spleen correlated to the magnitude of the immune response induced by pFMDV-GNP. In conclusion, we demonstrated the size-dependent immunogenic properties of pFMDV-GNP conjugates. Furthermore, we established that GNPs ranging from 8 to 17 nm in diameter may be ideal for eliciting a focused antibody response against a synthetic pFMDV peptide.
Assessment of the In Vivo Toxicity of Gold Nanoparticles
(2009-08-01)
Chen, Yu-Shiun
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Hung, Yao-Ching
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Liau, Ian
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Huang, G. Steve
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材料科學與工程學系
The environmental impact of nanoparticles is evident; however, their toxicity due to their nanosize is rarely discussed. Gold nanoparticles (GNPs) may serve as a promising model to address the size-dependent biological response to nanoparticles because they show good biocompatibility and their size can be controlled with great precision during their chemical synthesis. Naked GNPs ranging from 3 to 100 nm were injected intraperitoneally into BALB/C mice at a dose of 8 mg/kg/week. GNPs of 3, 5, 50, and 100 nm did not show harmful effects; however, GNPs ranging from 8 to 37 nm induced severe sickness in mice. Mice injected with GNPs in this range showed fatigue, loss of appetite, change of fur color, and weight loss. Starting from day 14, mice in this group exhibited a camel-like back and crooked spine. The majority of mice in these groups died within 21 days. Injection of 5 and 3 nm GNPs, however, did not induce sickness or lethality in mice. Pathological examination of the major organs of the mice in the diseased groups indicated an increase of Kupffer cells in the liver, loss of structural integrity in the lungs, and diffusion of white pulp in the spleen. The pathological abnormality was associated with the presence of gold particles at the diseased sites, which were verified by ex vivo Coherent anti-Stoke Raman scattering microscopy. Modifying the surface of the GNPs by incorporating immunogenic peptides ameliorated their toxicity. This reduction in the toxicity is associated with an increase in the ability to induce antibody response. The toxicity of GNPs may be a fundamental determinant of the environmental toxicity of nanoparticles.
Control of In Vivo Transport and Toxicity of Nanoparticles by Tea Melanin
(2012)
Chen, Yu-Shiun
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Hung, Yao-Ching
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Hong, Meng-Yeng
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Onischuk, Andrei Aleksandrovich
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Chiou, Jin Chern
"Nanoparticles are unfamiliar to researchers in toxicology. Toxicity may be generated simply due to the reduction in size. Compounds that prevent or cure toxic materials may not work on nanoparticles. Furthermore, as there are more and more applications of nanoparticles in drug delivery and in vivo imaging, controlling the transport and toxicity will be primary concerns for medical application of nanoparticles. Gold nanoparticles (GNPs) if injected intraperitoneally into mice can enter hippocampus and induce cognitive impairment. GNPs caused a global imbalance of monoamine levels, specifically affecting the dopaminergic and serotonergic neurons. Pretreatment of tea melanin significantly prevented the deposition of GNPs in mouse brains, especially in the hippocampus. Pretreatment of melanin completely alleviated GNP-induced impairment of cognition. Pre-administration of melanin stably maintained monoamines at normal profiles. Melanin completely prevented the invasion of GNPs into the Cornu Ammonis region of the hippocampus shown by coherent anti-Stoke Raman scattering microscopy. Here we show that the administration of tea melanin prevented the accumulation of Au in brain, the imbalance of monoamines, and the impairment of cognition in mice. The current study provides a therapeutic approach to toxicity of nanoparticles and a novel strategy to control the transport of GNP in mouse brain."
Detection of gold nanoparticles using an immunoglobulin-coated piezoelectric sensor
(2008-12-10)
Chen, Yu-Shiun
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Hung, Yao-Ching
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Chen, Kaochao
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Huang, Guewha Steven
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材料科學與工程學系
Since the existence of nanoparticles in our environment has already attracted considerable attention due to their possible toxic impact on biological systems, the field detection of nanoparticles is becoming a technology that will be much in need. We have constructed a piezoelectric sensor with an antibody-coated electrode. The antiserum can bind gold nanoparticles with a high degree of selectivity and sensitivity. The biosensor thus constructed can detect 4, 5, or 6 nm gold nanoparticles (GNPs) depending on the coated antiserum. The sensitivity for the detection of 5 nm GNPs was 10.3 +/- 0.9 ng Hz(-1), with the low limit of detection at 5.5 ng. A quartz crystal microbalance (QCM) sensor was capable of detecting GNPs and other types of nanoparticle, such as ZnO, or Fe(3)O(4). The current study provides, for the first time, a platform for detecting nanoparticles in a convenient, economical manner.
DNA sequencing using electrical conductance measurements of a DNA polymerase
(2013-06-01)
Chen, Yu-Shiun
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Lee, Chia-Hui
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Hung, Meng-Yen
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Pan, Hsu-An
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Chiou, Jin-Chern
The development of personalized medicine-in which medical treatment is customized to an individual on the basis of genetic information-requires techniques that can sequence DNA quickly and cheaply. Single-molecule sequencing technologies, such as nanopores, can potentially be used to sequence long strands of DNA without labels or amplification, but a viable technique has yet to be established. Here, we show that single DNA molecules can be sequenced by monitoring the electrical conductance of a phi29 DNA polymerase as it incorporates unlabelled nucleotides into a template strand of DNA. The conductance of the polymerase is measured by attaching it to a protein transistor that consists of an antibody molecule (immunoglobulin G) bound to two gold nanoparticles, which are in turn connected to source and drain electrodes. The electrical conductance of the DNA polymerase exhibits well-separated plateaux that are similar to 3 pA in height. Each plateau corresponds to an individual base and is formed at a rate of similar to 22 nucleotides per second. Additional spikes appear on top of the plateaux and can be used to discriminate between the four different nucleotides. We also show that the sequencing platform works with a variety of DNA polymerases and can sequence difficult templates such as homopolymers.
DNA sequencing using electrical conductance measurements of a DNA polymerase (Retracted article. See vol. 10, pg. 563, 2015)
(2013-06-01)
Chen, Yu-Shiun
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Lee, Chia-Hui
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Hung, Meng-Yen
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Pan, Hsu-An
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Chiou, Jin-Chern
The development of personalized medicine-in which medical treatment is customized to an individual on the basis of genetic information-requires techniques that can sequence DNA quickly and cheaply. Single-molecule sequencing technologies, such as nanopores, can potentially be used to sequence long strands of DNA without labels or amplification, but a viable technique has yet to be established. Here, we show that single DNA molecules can be sequenced by monitoring the electrical conductance of a phi29 DNA polymerase as it incorporates unlabelled nucleotides into a template strand of DNA. The conductance of the polymerase is measured by attaching it to a protein transistor that consists of an antibody molecule (immunoglobulin G) bound to two gold nanoparticles, which are in turn connected to source and drain electrodes. The electrical conductance of the DNA polymerase exhibits well-separated plateaux that are similar to 3 pA in height. Each plateau corresponds to an individual base and is formed at a rate of similar to 22 nucleotides per second. Additional spikes appear on top of the plateaux and can be used to discriminate between the four different nucleotides. We also show that the sequencing platform works with a variety of DNA polymerases and can sequence difficult templates such as homopolymers.
DNA sequencing using electrical conductance measurements of a DNA polymerase (Retracted article. See vol. 10, pg. 563, 2015)
(2013-06-01)
Chen, Yu-Shiun
;
Lee, Chia-Hui
;
Hung, Meng-Yen
;
Pan, Hsu-An
;
Chiou, Jin-Chern
The development of personalized medicine-in which medical treatment is customized to an individual on the basis of genetic information-requires techniques that can sequence DNA quickly and cheaply. Single-molecule sequencing technologies, such as nanopores, can potentially be used to sequence long strands of DNA without labels or amplification, but a viable technique has yet to be established. Here, we show that single DNA molecules can be sequenced by monitoring the electrical conductance of a phi29 DNA polymerase as it incorporates unlabelled nucleotides into a template strand of DNA. The conductance of the polymerase is measured by attaching it to a protein transistor that consists of an antibody molecule (immunoglobulin G) bound to two gold nanoparticles, which are in turn connected to source and drain electrodes. The electrical conductance of the DNA polymerase exhibits well-separated plateaux that are similar to 3 pA in height. Each plateau corresponds to an individual base and is formed at a rate of similar to 22 nucleotides per second. Additional spikes appear on top of the plateaux and can be used to discriminate between the four different nucleotides. We also show that the sequencing platform works with a variety of DNA polymerases and can sequence difficult templates such as homopolymers.
Gold nanoparticles caused learning impairment in mice
(2009)
Chen, Yu-Shiun
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Hung, Yao-Ching
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Liau, Ian
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Lin, Li-Wei
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Hong, Meng-Yeng
Aims : To investigate the toxic effect of gold nanoparticles (GNPs) to the learning and memory of mice. Main methods: Naked GNPs of 17 nm and 37 nm were synthesized, purified, and injected intraperitoneally into BALB/C mice at a dose of 8 mg/kg/week. Passive avoidance test was performed to detect the possible leaning impairment. Biochemical analysis to examine levels of monoamine and acetylcholine in mouse brain was also performed. ICP-MS was performed to examine the presence of GNP in brain. Localization of GNP at hippocampus was surveyed by ex vivo Coherent anti-Stoke Raman scattering (CARS) microscopy. Transmission electron microscopy (TEM) was also performed to verify the cellular location of GNPs at hippocampus. Key findings: Both 17 nm and 37 nm GNPs induced severe sickness in rnice. However, only 17 rim GNP impaired the learning and memory of mice. GNP treatment elevated levels of dopamine from 114.5 ng/mouse brain to 143.6 ng/mouse brain for 17 nm (p<0.01) and to 138.2 for 37 nm GNP (p<0.05). Serotinin was significantly reduced by 17 nm GNP treatment from 57.2 ng/mouse brain to 44.3 ng/mouse brain (p<0.05). ICP-MS indicated the presence of GNPs in every part of the brain. CARS microscopy showed that 17 nm GNP was located at the Cornu Amonis regions of hippocampus where neuronal cells clustered, while 37 nm GNP was excluded from the cell clustered region. TEM and EDAX verified the presence of 17 nm GNP in the cytoplasm and in the dendrite of pyramidal cell, while 37 rim GNP was found in the extracellular region of neuronal cells. TEM image also indicated that both endocytosis and free diffusion coexisted for the invasion of 17 nm GNP. Significance: The current study provided evidence that nanoparticles were capable of entering into brain and affecting normal brain function. The size-dependent invading ability of GNPs provides an extra dimension for drug delivery.
Gold nanoparticles regulate the blimp1/pax5 pathway and enhance antibody secretion in B-cells
(2014-03-28)
Lee, Chia-Hui
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Syu, Shih-Han
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Chen, Yu-Shiun
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Hussain, Saber M.
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Onischuk, Andrei Aleksandrovich
Nanoparticles are potential threats to human health and the environment; however, their medical applications as drug carriers targeting cancer cells bring hope to contemporary cancer therapy. As a model drug carrier, gold nanoparticles (GNPs) have been investigated extensively for in vivo toxicity. The effect of GNPs on the immune system, however, has rarely been examined. Antibody-secreting cells were treated with GNPs with diameters ranging from 2 to 50 nm. The GNPs enhanced IgG secretion in a size-dependent manner, with a peak of efficacy at 10 nm. The immune-stimulatory effect reached a maximum at 12 h after treatment but returned to control levels 24 h after treatment. This enhancing effect was validated ex vivo using B-cells isolated from mouse spleen. Evidence from RT-PCR and western blot experiments indicates that GNP-treatment upregulated B-lymphocyte-induced maturation protein 1 (blimp1) and downregulated paired box 5 (pax5). Immunostaining for blimp1 and pax5 in B-cells confirmed that the GNPs stimulated IgG secretion through the blimp1/pax5 pathway. The immunization of mice using peptide-conjugated GNPs indicated that the GNPs were capable of enhancing humoral immunity in a size-dependent manner. This effect was consistent with the bio-distribution of the GNPs in mouse spleen. In conclusion, in vitro, ex vivo, and in vivo evidence supports our hypothesis that GNPs enhance humoral immunity in mouse. The effect on the immune system should be taken into account if nanoparticles are used as carriers for drug delivery. In addition to their toxicity, the immune-stimulatory activity of nanoparticles could play an important role in human health and could have an environmental impact.
Measuring the flexibility of immunoglobulin by gold nanoparticles
(2006-11-08)
Huang, G. Steven
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Chen, Yu-Shiun
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Yeh, Hsiao-Wei
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材料科學與工程學系奈米科技碩博班
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奈米科技中心
We measured the flexibility of Fab and Fc arms of immunoglobulin using gold nanoparticles (GNPs). Enzyme-linked immunosorbent assay was performed to measure the affinity of anti-5 nm GNP antiserum against various sizes of GNPs. The flexibility of Fc was also measured by electron microscopy. The restricted binding affinity indicated that only a very limited amount of freedom was allowed for the Fab-Fab hinge, while Fab-Fc showed a much larger degree of freedom.
Picogram detection of metal ions by melanin-sensitized piezoelectric sensor
(2007-10-31)
Huang, G. Steven
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Wang, Meng-Te
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Su, Chia-Wei
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Chen, Yu-Shiun
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Hong, Meng-Yen
A heavy metal ion sensor was constructed by cross-linking melanin onto the gold electrode of quartz crystal microbalance (QCM). A mercury ion sensitivity of 518 +/- 37 Hz/ppm was observed, a substantial increase in sensitivity compared to previous reports of 10-50 Hz/ppm with the limit of detection at 5 ppb. Detection of other metal ions including Sn2+, Ge4+, Li+, Zn2+, Cu2+, Bi3+, Co2+, Al3+, Ni2+, Ag+, and Fe3+ were also performed. Unexpectedly, binding of Mn7+, Ph2+, Cd2+, and Cr3+ increased resonant frequencies. The surface profile of melanin thin film upon binding to metal ions was investigated by atomic force microscopy (AFM). Structural change of melanin upon binding to metal ions was characterized by circular dichroism and by infrared spectroscopy. The current study provides the first example of melanin-coated piezoelectric sensor showing high sensitivity and selectivity to metal ions. (c) 2007 Elsevier B.V. All rights reserved.
Self-assembly of an antireflective moth-like structure using antibody-functionalized nanowires and nanopore arrays
(2011-11-25)
Chen, Yu-Shiun
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Hung, Ning
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Chou, Li-Jen
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Chiou, Jin-Chern
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Hong, Meng-Yen
A novel framework to fabricate moth-like nanopillar arrays was proposed. In this scheme, nanowires were first cross-linked with anti-gold nanoparticle (GNP) antibodies and mixed with the nanopore array pre-deposited by GNP, which was then followed by centrifugation. An optimal success rate of 95% was finally obtained by choosing nanorods with an aspect ratio of 5:1 by modifying with 10 ng mL(-1) antibodies, and by inserting them into a pore array pre-deposited with 54.4 mu M GNP. The nanopillar arrays thus fabricated showed high levels of antireflective efficiency across a broad wavelength. Here we demonstrate the assembly of nanowires and nanopores into nanopillar arrays by the assistance of antibody-antigen binding. The application of bio-nano-interaction provides an economic, time-saving, and throughput approach to manipulating objects on the nanoscale.
Size-dependent impairment of cognition in mice caused by the injection of gold nanoparticles
(2010-12-03)
Chen, Yu-Shiun
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Hung, Yao-Ching
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Lin, Li-Wei
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Liau, Ian
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Hong, Meng-Yeng
We explored the size-dependent impairment of cognition in mice caused by the injection of gold nanoparticles (GNPs). GNPs of 17 and 37 nm in diameter were injected intraperitoneally into BALB/c mice at doses ranging from 0.5 to 14.6 mg kg(-1). ICP-MS was performed on brain tissue collected 1, 14 and 21 days after the injection. A passive-avoidance test was performed on day 21. Monoamine levels were determined on day 21. The microscopic distribution of GNPs in the hippocampus was examined using coherent anti-Stokes Raman scattering (CARS) microscopy and transmission electron microscopy (TEM). The results indicated that 17 nm GNPs passed through the blood-brain barrier more rapidly than 37 nm GNPs. Treatment with 17 nm GNPs decreased the latency time, which was comparable to the effect of scopolamine treatment, while 37 nm GNPs showed no significant effect. Dopamine levels and serotonin levels in the brain were significantly altered by the injection of 17 and 37 nm GNPs. GNPs affected dopaminergic and serotonergic neurons. CARS microscopy indicated that 17 nm GNPs entered the Cornu Ammonis (CA) region of the hippocampus, while 37 nm GNPs were excluded from the CA region. TEM verified the presence of 17 nm GNPs in the cytoplasm of pyramidal cells. In this study, we showed that the ability of GNPs to damage cognition in mice was size-dependent and associated with the ability of the particles to invade the hippocampus. The dosage and duration of the treatment should be taken into account if GNPs are used in the future as vehicles to carry therapeutic agents into the brain.
Ultrasensitive Detection of Cymbidium Mosaic Potexvirus Using a Single-Wall Carbon Nanotube-Functionalized Quartz Crystal Microbalance
(2010)
Chen, Yu-Shiun
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Hung, Yao-Ching
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Chiou, Jin-Chern
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Wang, Hui-Liang
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Huang, Hung-Shu
We have developed an ultrasensitive, convenient, real-time platform for detecting Cymbidium mosaic potexvirus (CymMV) based on single-wall carbon nanotube (SWNT)-functionalized quartz crystal microbalance (QCM) sensors. Functionalization was achieved by coating the QCM electrode with SWNTs, followed by 1,1'-carbonyldiimidazole-activated Tween 20 (CDI-Tween 20) modification and conjugation of antibodies. Sensitivity was enhanced from 2.18 to 11.5 Hz ng(-1) when 0.1 mu g mL(-1) CymMV was applied. The low limit of detection of SWNT-functionalized QCM sensors was improved from 2.08 to 0.502 ng. The SWNT-functionalized QCM sensor was successfully used to quantify the amount of CymMV contained in infected orchid leaves. Compared to enzyme-linked immunosorbent assay (ELISA), SWNT-functionalized QCM sensors are fast, economical, and ultra-sensitive, with comparable sensitivities. The current study demonstrates the application of QCM sensors as a convenient platform to detect and quantify CymMV. (C) 2010 The Japan Society of Applied Physics