Publications by authors named "Sang-Kyung Kim"

56 Publications

Highly Selective Multiplex Quantitative Polymerase Chain Reaction with a Nanomaterial Composite Hydrogel for Precise Diagnosis of Viral Infection.

ACS Appl Mater Interfaces 2021 Jul 24;13(26):30295-30305. Epub 2021 Jun 24.

Molecular Recognition Research Center, Biomedical Research Institute, Korea Institute of Science and Technology (KIST), Seoul 02792, Republic of Korea.

As viruses have been threatening global public health, fast diagnosis has been critical to effective disease management and control. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) is now widely used as the gold standard for detecting viruses. Although a multiplex assay is essential for identifying virus types and subtypes, the poor multiplicity of RT-qPCR makes it laborious and time-consuming. In this paper, we describe the development of a multiplex RT-qPCR platform with hydrogel microparticles acting as independent reactors in a single reaction. To build target-specific particles, target-specific primers and probes are integrated into the particles in the form of noncovalent composites with boron nitride nanotubes (BNNTs) and carbon nanotubes (CNTs). The thermal release characteristics of DNA, primer, and probe from the composites of primer-BNNT and probe-CNT allow primer and probe to be stored in particles during particle production and to be delivered into the reaction. In addition, BNNT did not absorb but preserved the fluorescent signal, while CNT protected the fluorophore of the probe from the free radicals present during particle production. Bicompartmental primer-incorporated network (bcPIN) particles were designed to harness the distinctive properties of two nanomaterials. The bcPIN particles showed a high RT-qPCR efficiency of over 90% and effective suppression of non-specific reactions. 16-plex RT-qPCR has been achieved simply by recruiting differently coded bcPIN particles for each target. As a proof of concept, multiplex one-step RT-qPCR was successfully demonstrated with a simple reaction protocol.
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http://dx.doi.org/10.1021/acsami.1c03434DOI Listing
July 2021

An NIR dual-emitting/absorbing inorganic compact pair: A self-calibrating LRET system for homogeneous virus detection.

Biosens Bioelectron 2021 Oct 29;190:113369. Epub 2021 May 29.

Molecular Recognition Research Center, Korea Institute of Science and Technology (KIST), Seoul, 02792, South Korea; Department of Chemistry, Hanyang University, Seoul, 04763, Republic of Korea. Electronic address:

Many conventional optical biosensing systems use a single responsive signal in the visible light region. This limits their practical applications, as the signal can be readily perturbed by various external environmental factors. Herein, a near-infrared (NIR)-based self-calibrating luminescence resonance energy transfer (LRET) system was developed for background-free detection of analytes in homogeneous sandwich-immunoassays. The inorganic LRET pair was comprised of NIR dual-emitting lanthanide-doped nanoparticles (LnNPs) as donors and NIR-absorbing LnNPs as acceptors, which showed a narrow absorption peak (800 nm) and long-term stability, enabling stable LRET with a built-in self-calibrating signal. Screened single-chain variable fragments (scFvs) were used as target avian influenza virus (AIV)-binding antibodies to increase the LRET efficiency in sandwich-immunoassays. The compact sensor platform successfully detected AIV nucleoproteins with a 0.38 pM limit of detection in buffer solution and 64 clinical samples. Hence, inorganic LnNP pairs may be effective for self-calibrating LRET systems in the background-free NIR region.
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http://dx.doi.org/10.1016/j.bios.2021.113369DOI Listing
October 2021

Highly sensitive and multiplexed one-step RT-qPCR for profiling genes involved in the circadian rhythm using microparticles.

Sci Rep 2021 Mar 19;11(1):6463. Epub 2021 Mar 19.

Center for Molecular Recognition Research, Materials and Life Science Research Division, Korea Institute of Science and Technology(KIST), Seoul, KS013, Korea.

Given the growing interest in molecular diagnosis, highly extensive and selective detection of genetic targets from a very limited amount of samples is in high demand. We demonstrated the highly sensitive and multiplexed one-step RT-qPCR platform for RNA analysis using microparticles as individual reactors. Those particles are equipped with a controlled release system of thermo-responsive materials, and are able to capture RNA targets inside. The particle-based assay can successfully quantify multiple target RNAs from only 200 pg of total RNA. The assay can also quantify target RNAs from a single cell with the aid of a pre-concentration process. We carried out 8-plex one-step RT-qPCR using tens of microparticles, which allowed extensive mRNA profiling. The circadian cycles were shown by the multiplex one-step RT-qPCR in human cell and human hair follicles. Reliable 24-plex one-step RT-qPCR was developed using a single operation in a PCR chip without any loss of performance (i.e., selectivity and sensitivity), even from a single hair. Many other disease-related transcripts can be monitored using this versatile platform. It can also be used non-invasively for samples obtained in clinics.
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http://dx.doi.org/10.1038/s41598-021-85728-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7979730PMC
March 2021

Cerium Oxide-Polysulfone Composite Separator for an Advanced Alkaline Electrolyzer.

Polymers (Basel) 2020 Nov 27;12(12). Epub 2020 Nov 27.

Hydrogen Research Department, Korea Institute of Energy Research (KIER), 152 Gajeong-ro, Yuseong-gu, Daejeon 34129, Korea.

The intermittent and volatile nature of renewable energy sources threatens the stable operation of power grids, necessitating dynamically operated energy storage. Power-to-gas technology is a promising method for managing electricity variations on a large gigawatt (GW) scale. The electrolyzer is a key component that can convert excess electricity into hydrogen with high flexibility. Recently, organic/inorganic composite separators have been widely used as diaphragm membranes; however, they are prone to increase ohmic resistance and gas crossover, which inhibit electrolyzer efficiency. Here, we show that the ceria nanoparticle and polysulfone composite separator exhibits a low area resistance of 0.16 Ω cm and a hydrogen permeability of 1.2 × 10 mol cm s bar in 30 wt% potassium hydroxide (KOH) electrolyte, which outperformed the commercial separator, the Zirfon PERL separator. The cell using a 100 nm ceria nanoparticle/polysulfone separator and advanced catalysts has a remarkable capability of 1.84 V at 800 mA cm at 30 wt% and 80 °C. The decrease in the average pore size of 77 nm and high wettability (contact angle 75°) contributed to the reduced ohmic resistance and low gas crossover. These results demonstrate that the use of ceria nanoparticle-based separators can achieve high performance compared to commercial zirconia-based separators.
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http://dx.doi.org/10.3390/polym12122821DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7759930PMC
November 2020

In-particle stem-loop RT-qPCR for specific and multiplex microRNA profiling.

Biosens Bioelectron 2020 Sep 18;163:112301. Epub 2020 May 18.

Center for Molecular Recognition Research, Materials and Life Science Research Division, Korea Institute of Science and Technology, Seoul, 02792, Republic of Korea; Biomedical Engineering, University of Science and Technology (UST), Daejeon, 34113, Republic of Korea. Electronic address:

Here we report a novel method of microRNA (miRNA) profiling with particle-based multiplex quantitative reverse transcription polymerase chain reaction (RT-qPCR). To achieve target-specific reaction in a particle, the stem-loop RT primer and forward primer for each target miRNA were chemically immobilized to the particle. Target-specific cDNA synthesis proceeds with the stem-loop RT primer and then qPCR subsequently proceeds with the forward primer to rapidly achieve a quantitative result. High-fidelity multiplex assay was also accomplished in a single PCR process by loading multiple particles for each specific miRNA. The method for primer supply in the particles, involving confinement of the target-specific RT and PCR primers in the matrix of particles, led to the reduction of nonspecific reactions and improved the selectivity of the miRNA assay while minimizing labor in a multiple target assay. Specifically, this particle-based assay enabled the differentiation of mature miRNA from precursor with selectivity of 270:1 in terms of amplification speed. This advanced method also showed good discrimination among highly homologous let-7 family members, with cross-reaction rates of less than 5%. We demonstrated a very simple process of five-plex miRNA profiling in total RNA, and the measured changes in expression level were consistent with those from a conventional singleplex method.
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http://dx.doi.org/10.1016/j.bios.2020.112301DOI Listing
September 2020

Thermo-Responsive Polymer Capsules in Real-Time One-Step RT-PCR for Highly Multiplex RNA Analysis.

Adv Healthc Mater 2020 04 5;9(7):e1900790. Epub 2020 Mar 5.

Center for Molecular Recognition Research, Materials and Life Science Research Division, Korea Institute of Science and Technology, Seoul, 02792, Republic of Korea.

Rapid and simple detection of RNA targets is in high demand due to the growing threat of pandemic viruses. One-step real-time, reverse transcription-polymerase chain reaction (One-step RT-qPCR) using a controlled release system of thermo-responsive materials is developed in this paper to enable high-fidelity RNA analysis as suppressing by-products. The nanocapsules, consisting of upper critical solution temperature (UCST) material and PCR primers, carry or release the primers depending upon the temperature. The UCST nanocapsules are introduced into hydrogel microparticles incorporated with RT primers and then the target RNA is selectively amplified in the microparticle through one-step RT-qPCR. Severe side products are sharply subdued by separating the PCR primers from the RT process by means of the microparticles with nanocapsules. Because the one-step assay is now implemented in a single microparticle, multiple target RNAs can be analyzed in a simple RT-qPCR of multiple particles. Reliable 18-plex one-step RT-qPCR is successfully conducted within 30 min using single-color fluorescent optics. This work also explains the facile fabrication processes used for the thermo-responsive nanocapsules and hydrogel microparticles by the blending polymerization method. Extensible multiplex analysis of influenza virus demonstrates the versatile uses of this one-step RT-qPCR platform.
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http://dx.doi.org/10.1002/adhm.201900790DOI Listing
April 2020

A Capacitive Micromachined Ultrasonic Transducer-Based Resonant Sensor Array for Portable Volatile Organic Compound Detection with Wireless Systems.

Sensors (Basel) 2019 Mar 21;19(6). Epub 2019 Mar 21.

School of Electrical Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Korea.

The development of portable volatile organic compound (VOC) sensors is essential for home healthcare and workplace safety because VOCs are environmental pollutants that may critically affect human health. Here, we report a compact and portable sensor platform based on a capacitive micromachined ultrasonic transducer (CMUT) array offering multiplex detection of various VOCs (toluene, acetone, ethanol, and methanol) using a single read-out system. Three CMUT resonant devices were functionalized with three different layers: (1) phenyl-selective peptide, (2) colloids of single-walled nanotubes and peptide, and (3) poly(styrene-co-allyl alcohol). As each device exhibited different sensitivities to the four VOCs, we performed principal component analysis to achieve selective detection of all four gases. For the simultaneous detection of VOCs using CMUT sensors, the changes in the resonant frequencies of three devices were monitored in real time, but using only a single oscillator through an electrically controlled relay to achieve compactness. In addition, by devising a wireless system, measurement results were transmitted to a smartphone to monitor the concentration of VOCs. We used multiple sensors to obtain a larger number of fingerprints for pattern recognition to enhance selectivity but interfaced these sensors with a single read-out circuit to minimize the footprint of the overall system. The compact CMUT-based sensor array based on a multiplex detection scheme is a promising sensor platform for portable VOC monitoring.
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http://dx.doi.org/10.3390/s19061401DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6470568PMC
March 2019

Fully Packaged Portable Thin Film Biosensor for the Direct Detection of Highly Pathogenic Viruses from On-Site Samples.

ACS Nano 2019 01 31;13(1):812-820. Epub 2018 Dec 31.

Center for Biomaterials , Korea Institute of Science and Technology (KIST) , Seoul 02792 , Republic of Korea.

The thin film transistor (TFT) is a promising biosensor system with great sensitivity, label-free detection, and a quick response time. However, even though the TFT sensor has such advantageous characteristics, the disadvantages hamper the TFT sensor's application in the clinical field. The TFT is susceptible to light, noise, vibration, and limited usage, and this significantly limits its on-site potential as a practical biosensor. Herein, we developed a fully packaged, portable TFT electrochemical biosensor into a chip form, providing both portability through minimizing the laboratory equipment size and multiple safe usages by protecting the semiconductor sensor. Additionally, a safe environment that serves as a miniature probe station minimizes the previously mentioned disadvantages, while providing the means to properly link the TFT biosensor with a portable analyzer. The biosensor was taken into a biosafety level 3 (BSL-3) laboratory setting to analyze highly pathogenic avian influenza virus (HPAIV) samples. This virus quickly accumulates within a host, and therefore, early stage detection is critical to deterring the further spread of the deadly disease to other areas. However, current on-site methods have poor limits of detection (10-10 EID/mL), and because the virus has low concentration in its early stages, it cannot be detected easily. We have compared the sample measurements from our device with virus concentration data obtained from a RT-PCR (virus range: 10-10 EID/mL) and have identified an increasing voltage signal which corresponds to increasing virus concentration.
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http://dx.doi.org/10.1021/acsnano.8b08298DOI Listing
January 2019

Zygotic gene activation in the chicken occurs in two waves, the first involving only maternally derived genes.

Elife 2018 10 30;7. Epub 2018 Oct 30.

Department of Agricultural Biotechnology, Seoul National University, Seoul, Republic of Korea.

The first wave of transcriptional activation occurs after fertilisation in a species-specific pattern. Despite its importance to initial embryonic development, the characteristics of transcription following fertilisation are poorly understood in Aves. Here, we report detailed insights into the onset of genome activation in chickens. We established that two waves of transcriptional activation occurred, one shortly after fertilisation and another at Eyal-Giladi and Kochav Stage V. We found 1544 single nucleotide polymorphisms across 424 transcripts derived from parents that were expressed in offspring during the early embryonic stages. Surprisingly, only the maternal genome was activated in the zygote, and the paternal genome remained silent until the second-wave, regardless of the presence of a paternal pronucleus or supernumerary sperm in the egg. The identified maternal genes involved in cleavage that were replaced by bi-allelic expression. The results demonstrate that only maternal alleles are activated in the chicken zygote upon fertilisation, which could be essential for early embryogenesis and evolutionary outcomes in birds.
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http://dx.doi.org/10.7554/eLife.39381DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6242549PMC
October 2018

Expression of transcription factors during area pellucida formation in intrauterine chicken embryos.

Int J Dev Biol 2018 ;62(4-5):341-345

Department of Agricultural Biotechnology, and Research Institute of Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University, Seoul, Korea.

Initial embryological development in avian species, consisting of cleavage and area pellucida formation, occurs prior to oviposition. In chickens, the first lineage segregation is known to occur during the last 10 hours of intrauterine development, a finding which has primarily been identified on the basis of morphological perspectives. We traced the early expression of the transcription factors NANOG, POUV and EOMES at Eyal-Giladi and Kochav (EGK) stages VI through X using in situ hybridization. At EGK.VI, NANOG and EOMES were heterogeneously expressed in a salt-and-pepper manner. From EGK.VIII to EGK.X, NANOG- or EOMES-positive cells were predominantly located in the epiblast or area opaca regions, respectively. POUV-expressing cells were found in the upper layer at EGK.VIII. After oviposition, POUV mRNA was strongly expressed in the epiblast, but weakly expressed in the hypoblast at EGK.X. Furthermore, NANOG- and POUV-negative cells were located in the subgerminal cavity where layer reduction occurs during area pellucida formation. Those cells were larger and did not seem to contribute to epithelialization until EGK.X. Our results on the spatiotemporal expression of transcription factors contribute to a greater understanding of the dynamic process of intrauterine development in chickens.
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http://dx.doi.org/10.1387/ijdb.170255jhDOI Listing
April 2019

Rapid and background-free detection of avian influenza virus in opaque sample using NIR-to-NIR upconversion nanoparticle-based lateral flow immunoassay platform.

Biosens Bioelectron 2018 Jul 22;112:209-215. Epub 2018 Apr 22.

Molecular Recognition Research Center, Korea Institute of Science and Technology (KIST), Division of Nano and Information Technology, KIST School, Korea University of Science and Technology (UST), Seoul 02792, South Korea. Electronic address:

Rapid and sensitive on-site detection of avian influenza virus (AIV) is the key for achieving near real-time surveillance of AIV and reducing the risk of dissemination. However, unlike the laboratory-prepared transparent buffer solutions containing a single type of influenza virus, distinction between real- and false- positive outputs and detection of low concentrations of AIV in stool specimens or cloacal swabs are difficult. Here, we developed a rapid and background-free lateral flow immunoassay (LFA) platform that utilizes near-infrared (NIR)-to-NIR upconversion nanoparticles (UCNPs) to yield a sensor that detects AIV nucleoproteins (NPs) from clinical samples within 20 min. Ca as a heterogeneous dopant ion in the shell enhanced the NIR-to-NIR upconversion photoluminescence (PL) emission without inducing significant changes in the morphology of the UCNPs. In a mixture of opaque stool samples and gold nanoparticles (GNPs), which are components of commercial AIV LFA, the background signal of the stool samples masked the absorption peak of GNPs. However, UCNPs dispersed in the stool samples still show strong emission centered at 800 nm when excited at 980 nm, which enables the NIR-to-NIR upconversion nanoparticle-based lateral flow immunoassay (NNLFA) platform to detect 10-times lower viral load than a commercial GNP-based AIV LFA. The detection limit of NNLFA for LPAI H5N2 and HPAI H5N6 viruses was 10 and 10 EID/mL, respectively. Moreover, the viruses were successfully detected within dark brown-colored samples using the NNLFA but not the commercial AIV LFA. Therefore, the rapid and background-free NNLFA platform can be used for sensitive on-site detection of AIV.
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http://dx.doi.org/10.1016/j.bios.2018.04.047DOI Listing
July 2018

The first whole transcriptomic exploration of pre-oviposited early chicken embryos using single and bulked embryonic RNA-sequencing.

Gigascience 2018 04;7(4):1-9

Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Korea.

Background: The chicken is a valuable model organism, especially in evolutionary and embryology research because its embryonic development occurs in the egg. However, despite its scientific importance, no transcriptome data have been generated for deciphering the early developmental stages of the chicken because of practical and technical constraints in accessing pre-oviposited embryos.

Findings: Here, we determine the entire transcriptome of pre-oviposited avian embryos, including oocyte, zygote, and intrauterine embryos from Eyal-giladi and Kochav stage I (EGK.I) to EGK.X collected using a noninvasive approach for the first time. We also compare RNA-sequencing data obtained using a bulked embryo sequencing and single embryo/cell sequencing technique. The raw sequencing data were preprocessed with two genome builds, Galgal4 and Galgal5, and the expression of 17,108 and 26,102 genes was quantified in the respective builds. There were some differences between the two techniques, as well as between the two genome builds, and these were affected by the emergence of long intergenic noncoding RNA annotations.

Conclusion: The first transcriptome datasets of pre-oviposited early chicken embryos based on bulked and single embryo sequencing techniques will serve as a valuable resource for investigating early avian embryogenesis, for comparative studies among vertebrates, and for novel gene annotation in the chicken genome.
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http://dx.doi.org/10.1093/gigascience/giy030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5893961PMC
April 2018

Acquisition of pluripotency in the chick embryo occurs during intrauterine embryonic development via a unique transcriptional network.

J Anim Sci Biotechnol 2018 10;9:31. Epub 2018 Apr 10.

1Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University, Seoul, 08826 Korea.

Background: Acquisition of pluripotency by transcriptional regulatory factors is an initial developmental event that is required for regulation of cell fate and lineage specification during early embryonic development. The evolutionarily conserved core transcriptional factors regulating the pluripotency network in fishes, amphibians, and mammals have been elucidated. There are also species-specific maternally inherited transcriptional factors and their intricate transcriptional networks important in the acquisition of pluripotency. In avian species, however, the core transcriptional network that governs the acquisition of pluripotency during early embryonic development is not well understood.

Results: We found that chicken () was expressed in the stages between the pre-ovulatory follicle and oocyte and was continuously detected in Eyal-Giladi and Kochav stage I (EGK.I) to X. However, was not expressed during folliculogenesis, but began to be detectable between EGK.V and VI. Unexpectedly, could not be detected during folliculogenesis and intrauterine embryonic development. Instead of , was maternally inherited and continuously expressed during chicken intrauterine development. In addition, we found that the pluripotency-related genes such as , , , , , and began to be dramatically upregulated between EGK.VI and VIII.

Conclusion: These results suggest that chickens have a unique pluripotent circuitry since maternally inherited c and may play an important role in the initial acquisition of pluripotency. Moreover, the acquisition of pluripotency in chicken embryos occurs at around EGK.VI to VIII.
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http://dx.doi.org/10.1186/s40104-018-0246-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5891889PMC
April 2018

The transgenic chicken derived anti-CD20 monoclonal antibodies exhibits greater anti-cancer therapeutic potential with enhanced Fc effector functions.

Biomaterials 2018 06 13;167:58-68. Epub 2018 Mar 13.

Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University, Seoul, 08826, South Korea; Institute for Biomedical Sciences, Shinshu University, Minamiminowa, Nagano, 399-4598, Japan. Electronic address:

Modern genetic techniques, enable the use of animal bioreactor systems for the production and functional enhancement of anti-cancer antibodies. Chicken is the most efficient animal bioreactor for the production of anti-cancer antibodies because of its relatively short generation time, plentiful reproductive capacity, and daily deposition in the egg white. Although several studies have focused on the production of anti-cancer antibodies in egg white, in-depth studies of the biological activity and physiological characteristics of transgenic chicken-derived anti-cancer antibodies have not been fully carried out. Here, we report the production of an anti-cancer monoclonal antibody against the CD20 protein from egg whites of transgenic hens, and validated the bio-functional activity of the protein in B-lymphoma and B-lymphoblast cells. Quantitative analysis showed that deposition of the chickenised CD20 monoclonal antibody (cCD20 mAb) from transgenic chickens increased in successive generations and with increasing transgene copy number. Ultra-performance liquid chromatography (UPLC) tandem mass spectrometry (LC/MS/MS) analysis showed that the cCD20 mAb exhibited 14 N-glycan patterns with high-mannose, afucosylation and terminal galactosylation. The cCD20 mAb did not exhibit significantly improved Fab-binding affinity, but showed markedly enhanced Fc-related functions, including complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) compared to commercial rituximab, a chimeric mAb against CD20. Our results suggest that the transgenic chicken bioreactor is an efficient system for producing anti-cancer therapeutic antibodies with enhanced Fc effector functions.
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http://dx.doi.org/10.1016/j.biomaterials.2018.03.021DOI Listing
June 2018

The transcriptome of early chicken embryos reveals signaling pathways governing rapid asymmetric cellularization and lineage segregation.

Development 2018 03 14;145(6). Epub 2018 Mar 14.

Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Korea

The phylogenomics and comparative functional genomics of avian species were investigated in the Bird 10,000 Genomes (B10K) project because of the important evolutionary position of birds and their value as a research model. However, the systematic profiling of transcriptional changes prior to oviposition has not been investigated in avian species because of the practical difficulties in obtaining pre-oviposited eggs. In this study, a total of 137 pre-oviposited embryos were collected from hen ovaries and oviducts and subjected to RNA-sequencing analyses. Two waves of chicken zygotic genome activation (ZGA) were observed. Functionally distinct developmental programs involving Notch, MAPK, Wnt and TGFβ signaling were separately detected during cleavage and area pellucida formation. Furthermore, the early stages of chicken development were compared with the human and mouse counterparts, highlighting chicken-specific signaling pathways and gradually analogous gene expression via ZGA. These findings provide a genome-wide understanding of avian embryogenesis and comparisons among amniotes.
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http://dx.doi.org/10.1242/dev.157453DOI Listing
March 2018

Multiplex real-time PCR using temperature sensitive primer-supplying hydrogel particles and its application for malaria species identification.

PLoS One 2018 2;13(1):e0190451. Epub 2018 Jan 2.

Center for BioMicroSystems, Brain Science Institute, Korea Institute of Science and Technology (KIST), Seoul, Seongbuk-gu, Korea.

Real-time PCR, also called quantitative PCR (qPCR), has been powerful analytical tool for detection of nucleic acids since it developed. Not only for biological research but also for diagnostic needs, qPCR technique requires capacity to detect multiple genes in recent years. Solid phase PCR (SP-PCR) where one or two directional primers are immobilized on solid substrates could analyze multiplex genetic targets. However, conventional SP-PCR was subjected to restriction of application for lack of PCR efficiency and quantitative resolution. Here we introduce an advanced qPCR with primer-incorporated network (PIN). One directional primers are immobilized in the porous hydrogel particle by covalent bond and the other direction of primers are temporarily immobilized at so-called 'Supplimers'. Supplimers released the primers to aqueous phase in the hydrogel at the thermal cycling of PCR. It induced the high PCR efficiency over 92% with high reliability. It reduced the formation of primer dimers and improved the selectivity of qPCR thanks to the strategy of 'right primers supplied to right place only'. By conducting a six-plex qPCR of 30 minutes, we analyzed DNA samples originated from malaria patients and successfully identified malaria species in a single reaction.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0190451PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5749795PMC
February 2018

Hydrogel micropost-based qPCR for multiplex detection of miRNAs associated with Alzheimer's disease.

Biosens Bioelectron 2018 Mar 19;101:235-244. Epub 2017 Oct 19.

Center for BioMicrosystems, Brain Science Institute, Korea Institute of Science and Technology (KIST), Seoul 02792, Republic of Korea; Division of Bio-Medical Science & Technology, KIST School, Korea University of Science and Technology (UST), Seoul 02792, Republic of Korea. Electronic address:

Quantitative polymerase chain reaction (qPCR) renders profiling of genes of interest less time-consuming and cost-effective. Recently, multiplex profiling of miRNAs has enabled identifying or investigating predominant miRNAs for various diseases such as cancers and neurodegenerative diseases. Conventional multiplex qPCR technologies mostly use colorimetric measurements in solution phase, yet not only suffer from limited multiplexing capacity but also require target-screening processes due to non-specific binding between targets and primers. Here, we present hydrogel micropost-based qPCR for multiplex detection of miRNAs associated with Alzheimer's disease (AD). Our methodology promises two key advantages compared with the conventional solution-based PCR: 1) nearly no non-specific crosstalks between targets and primers, and 2) practically valuable multiplexing by spatial encoding within a single microchamber. Specifically, we immobilized hydrogel microposts (~ 400µm in diameter) within commercially available polycarbonate PCR chips by multi-step ultraviolet (UV, 365nm) exposure. We optimized this photoimmobilization for thermal cycles of PCR as well. Acrylated forward primers incorporated in polyethylene glycol diacrylate (PEGDA) posts played a crucial role to confine fluorescent signal of cDNA amplification within the PEGDA hydrogel. To demonstrate the potential of our platform, we successfully verified multiplex detection of five miRNAs, which were reported to be highly correlated with AD, from a complex buffer of human plasma.
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http://dx.doi.org/10.1016/j.bios.2017.10.039DOI Listing
March 2018

Multiplex Real-Time PCR Using Encoded Microparticles for MicroRNA Profiling.

Methods Mol Biol 2017 ;1654:221-230

Center for BioMicrosystems, Brain Science Institute, Korea Institute of Science and Technology, Seoul, South Korea.

Multiplex quantitative real-time PCR (qPCR), which measures multiple DNAs in a given sample, has drawn unprecedented attention as a means of verifying the rapidly increasing genetic targets in a single phenotype. We report the detailed procedure of a readily extensible qPCR for multiple microRNA (miRNA) expression analysis using microparticles of primer-immobilized networks as discrete reactors. Individual particles are identified by two-dimensional codes engraved into the particles. It allows high-fidelity signal analysis in the multiplex real-time PCR. During the course of PCR, the amplicons accumulate in the volume of the particles with amplification efficiency over 95%. Tens of miRNAs can be quantitatively profiled in a single PCR reaction of this method.
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http://dx.doi.org/10.1007/978-1-4939-7231-9_15DOI Listing
June 2018

Aptamer-Based Single-Step Assay by the Fluorescence Enhancement on Electroless Plated Nano Au Substrate.

Sensors (Basel) 2017 Sep 7;17(9). Epub 2017 Sep 7.

Center for BioMicrosystems, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Sungbuk-gu, Seoul 136-791, Korea.

A new single-step aptamer-based surface-enhanced fluorescent optical sensor is built, by combining an aptamer-target interaction for target recognition and a fluorophore interaction for signal enhancement. The developed aptasensor is simple, sensitive, specific and stable for the detection of thrombin. A new nanometallic Au structure in the range of 100 nm was constructed through effective electroless plating method on a Cu thin film. Cu⁺ ions act as sacrificial seeds for the reduction of Au ions to form Au nanolawns. In order to utilize the structure for a fluorescence-based sensor, aptamer conjugated with Cy3 was immobilized on the nanogold substrate through electrostatic attraction. The Au substrate was coated with chitosan (molecular weight 1000 Da). Thrombin binding aptamer (TBA) was applied as a model system demonstrating the aptamer-based fluorescence assay on nanogold substrates. Thrice-enhanced fluorescence emission was achieved with Cy3-conjugated TBA stably immobilized on the chitosan-coated Au substrate. The intensity change was proportional to the concentration of thrombin from 10 μM to 10 pM, whereas the intensity change was ignorable for other proteins such as human serum albumin (HSA). Aptamer-based assay benefited from simple immobilization of receptors and Au nanostructure contributed in building an effective surface enhancing/positively charged substrate was proved. Such an aptasensor holding high utilities for point-of-care devices by incorporating simplicity, sensitivity and selectivity in detection, low-cost for test, small sample volumes has been developed.
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http://dx.doi.org/10.3390/s17092044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5620733PMC
September 2017

Detection of Avian Influenza Virus from Cloacal Swabs Using a Disposable Well Gate FET Sensor.

Adv Healthc Mater 2017 Jul 16;6(13). Epub 2017 May 16.

Center for Biomaterials, Biomedical Research Institute, Korea Institute of Science and Technology (KIST), 5 Hwarang-ro 14-gil, Seongbuk-gu, Seoul, 02792, Republic of Korea.

Current methods to detect avian influenza viruses (AIV) are time consuming and lo inw sensitivity, necessitating a faster and more sensitive sensor for on-site epidemic detection in poultry farms and urban population centers. This study reports a field effect transistor (FET) based AIV sensor that detects nucleoproteins (NP) within 30 minutes, down to an LOD of 10 EID mL from a live animal cloacal swab. Previously reported FET sensors for AIV detection have not targeted NPs, an internal protein shared across multiple strains, due to the difficulty of field-effect sensing in a highly ionic lysis buffer. The AIV sensor overcomes the sensitivity limit with an FET-based platform enhanced with a disposable well gate (DWG) that is readily replaceable after each measurement. In a single procedure, the virus-containing sample is immersed in a lysis buffer mixture to expose NPs to the DWG surface. In comparison with commercial AIV rapid kits, the AIV sensor is proved to be highly sensitive, fast, and compact, proving its potential effectiveness as a portable biosensor.
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http://dx.doi.org/10.1002/adhm.201700371DOI Listing
July 2017

Extensible multiplex real-time PCR for rapid bacterial identification with carbon nanotube composite microparticles.

Biosens Bioelectron 2017 Aug 1;94:256-262. Epub 2017 Mar 1.

Center for BioMicrosystems, Brain Science Institute, Korea Institute of Science and Technology (KIST), Seoul 02792, Republic of Korea; Department of Biomedical Engineering, Korea University of Science and Technology (UST), Daejeon 34113, Republic of Korea. Electronic address:

The early diagnosis of pathogenic bacteria is significant for bacterial identification and antibiotic resistance. Implementing rapid, sensitive, and specific detection, molecular diagnosis has been considered complementary to the conventional bacterial culture. Composite microparticles of a primer-immobilized network (cPIN) are developed for multiplex detection of pathogenic bacteria with real-time polymerase chain reaction (qPCR). A pair of specific primers are incorporated and stably conserved in a cPIN particle. One primer is crosslinked to the polymer network, and the other is bound to carbon nanotubes (CNTs) in the particle. At the initiation of qPCR, the latter primer is released from the CNTs and participates in the amplification. The amplification efficiency of this cPIN qPCR is estimated at more than 90% with suppressed non-specific signals from complex samples. In multiplexing, four infective pathogens are successfully discriminated using this cPIN qPCR. Multiplex qPCR conforms with the corresponding singleplex assays, proving independent amplification in each particle. Four bacterial targets from clinical samples are differentially analyzed in 30min of a single qPCR trial with multiple cPIN particles.
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http://dx.doi.org/10.1016/j.bios.2017.02.049DOI Listing
August 2017

Selective and vertical microfabrication of lipid tubule arrays on glass substrates using template-guided gentle hydration.

Lab Chip 2016 11;16(24):4732-4741

Center for BioMicrosystems, Brain Science Institute, Korea Institute of Science and Technology, Seoul 02792, Republic of Korea. and Department of Biomedical Engineering, University of Science and Technology, Daejeon 34113, Republic of Korea.

Generally, asymmetric tubular lipid structures have been formed under the specific condition of gentle hydration or by using hydrodynamic and/or electrical elongation of vesicular lipid structures. Small-size lipid tubes are, however, very difficult to allocate or align in the vertical direction on the specific site of the substrate and, therefore, the ability to produce them selectively and in large quantities as an array form is limited. Herein, we propose an easy and novel method to fabricate selective and vertical lipid tube arrays using template-guided gentle hydration of dried lipid films without any external forces. A lipid solution was drop-dispensed onto a porous membrane and dried to form a lipid film. Then, the lipid-coated porous membrane was transferred to a glass substrate by using a UV-cured polymer layer to achieve tight bonding. Upon swelling with an appropriate buffer, expansion forces due to osmotic pressure during the gentle hydration process were highly constrained to confined pores, thereby resulting in the nucleation of tube-like lipid structures through the pores. Interestingly, according to the aspect ratio of pores (AR, pore length/pore diameter), different shapes of lipid structures, including vesicular, oval, and tube-like, were generated, which indicates the importance of the AR, as well as the pore diameter, during fabrication of tubular lipid structures. Also, this approach was easily modified with 1% chitosan to enhance the stability of the lipid tubes (>30 min in life time), by lipid coating twice and by using unsaturated lipids to increase tube length (>30 μm in length). Therefore, in the future, the simple but robust template-guided gentle hydration method will be a useful tool for fabricating addressable and engineered lipid tube arrays as a sensory unit.
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http://dx.doi.org/10.1039/c6lc01095dDOI Listing
November 2016

Microparticle-based RT-qPCR for highly selective rare mutation detection.

Biosens Bioelectron 2017 Jan 18;87:229-235. Epub 2016 Aug 18.

Center for BioMicrosystems, Brain Science Institute, Korea Institute of Science and Technology, Seoul 02792, Republic of Korea; Department of Biomedical Engineering, Korea University of Science and Technology, Daejeon 34113, Republic of Korea. Electronic address:

The quantitative reverse transcription polymerase chain reaction (RT-qPCR) has become one of the most widely used methods in the detection of disease-specific RNAs. The RT-qPCR involves two separate steps, RT and qPCR. In this study, we suggest a new RT-qPCR protocol with the particles of primer-immobilized networks (PINs), performing capture, RT and amplification of a target RNA in one particle. The production of undesired cDNAs was dramatically suppressed by the specific capture of the target RNA within the particle. Afterward, RT and amplification processes are performed without loss of cDNAs as exchanging the reaction solution. The biomarker gene of chronic myeloid leukemia, Bcr-Abl fusion transcript, is detected in the sensitivity of single mutant leukemic cell mixed in 10 normal cell using this protocol with the excellent restraint of non-specific signal. This protocol that whole processes are performed in the particle in a row is preferred for the highly specific detection of target RNAs in complex sample.
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http://dx.doi.org/10.1016/j.bios.2016.08.057DOI Listing
January 2017

Highly sensitive sandwich-type SPR based detection of whole H5Nx viruses using a pair of aptamers.

Biosens Bioelectron 2016 Dec 21;86:293-300. Epub 2016 Jun 21.

Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Anam-dong, Seongbuk-gu, Seoul 136-713, Republic of Korea. Electronic address:

In this research, we report highly sensitive and specific sandwich-type SPR-based biosensor for the detection H5Nx whole viruses. A few of aptamers, for the first time, were successfully screened and characterized for whole avian influenza (AI) viruses, H5Nx, by using Multi-GO-SELEX method. The affinities of the aptamers developed in this study were ranged from 8×10(4) to 1×10(4)EID50/ml, and the aptamers IF22, IF23 were found to be specific to H5N1 and H5N8, respectively. In addition, some flexible aptamers IF20, IF15, and IF10 were found to bind to the H5N1 and H5N2, H5N1 and H5N8, or H5N1, H5N2, and H5N8, respectively. Moreover, aptamers IF10 and IF22 were found to bind H5N1 virus simultaneously and confirmed to bind the different site of the same H5N1 whole virus. Therefore, this pair of aptamers, IF10 and IF22, were successfully applied to develop the sandwich-type SPR-based biosensor assay which is rapid, accurate for the detection of AI whole virus from H5N1-infected feces samples. The minimum detectible concentration of H5N1 whole virus was found to be 200 EID50/ml with this sandwich-type detection using the aptamer pair obtained in this study. In addition, the sensitivity of this biosensor was successfully enhanced by using the signal amplification with the secondary aptamer conjugated with gold nanoparticles.
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http://dx.doi.org/10.1016/j.bios.2016.06.064DOI Listing
December 2016

Extensible Multiplex Real-time PCR of MicroRNA Using Microparticles.

Sci Rep 2016 Mar 11;6:22975. Epub 2016 Mar 11.

Center for BioMicrosystems, Brain Science Institute, Korea Institute of Science and Technology (KIST), Seoul, Korea.

Multiplex quantitative real-time PCR (qPCR), which measures multiple DNAs in a given sample, has received significant attention as a mean of verifying the rapidly increasing genetic targets of interest in single phenotype. Here we suggest a readily extensible qPCR for the expression analysis of multiple microRNA (miRNA) targets using microparticles of primer-immobilized networks as discrete reactors. Individual particles, 200~500 μm in diameter, are identified by two-dimensional codes engraved into the particles and the non-fluorescent encoding allows high-fidelity acquisition of signal in real-time PCR. During the course of PCR, the amplicons accumulate in the volume of the particles with high reliability and amplification efficiency over 95%. In a quick assay comprising of tens of particles holding different primers, each particle brings the independent real-time amplification curve representing the quantitative information of each target. Limited amount of sample was analyzed simultaneously in single chamber through this highly multiplexed qPCR; 10 kinds of miRNAs from purified extracellular vesicles (EVs).
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http://dx.doi.org/10.1038/srep22975DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4786821PMC
March 2016

Evaluation of Chemical Interactions between Small Molecules in the Gas Phase Using Chemical Force Microscopy.

Sensors (Basel) 2015 Dec 4;15(12):30683-92. Epub 2015 Dec 4.

Center for Biomicrosystems, Korea Institute of Science and Technology (KIST), Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791, Korea.

Chemical force microscopy analyzes the interactions between various chemical/biochemical moieties in situ. In this work we examined force-distance curves and lateral force to measure the interaction between modified AFM tips and differently functionalized molecular monolayers. Especially for the measurements in gas phase, we investigated the effect of humidity on the analysis of force-distance curves and the images in lateral force mode. Flat chemical patterns composed of different functional groups were made through micro-contact printing and lateral force mode provided more resolved analysis of the chemical patterns. From the images of 1-octadecanethiol/11-mercapto-1-undecanoic acid patterns, the amine group functionalized tip brought out higher contrast of the patterns than an intact silicon nitride tip owing to the additional chemical interaction between carboxyl and amine groups. For more complex chemical interactions, relative chemical affinities toward specific peptides were assessed on the pattern of 1-octadecanethiol/phenyl-terminated alkanethiol. The lateral image of chemical force microscopy reflected specific preference of a peptide to phenyl group as well as the hydrophobic interaction.
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http://dx.doi.org/10.3390/s151229823DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4721743PMC
December 2015

Single-carbon discrimination by selected peptides for individual detection of volatile organic compounds.

Sci Rep 2015 Mar 17;5:9196. Epub 2015 Mar 17.

Center for Spintronics, Post-Silicon Semiconductor Institute, Korea Institute of Science and Technology, Seoul. 136-791, Republic of Korea.

Although volatile organic compounds (VOCs) are becoming increasingly recognized as harmful agents and potential biomarkers, selective detection of the organic targets remains a tremendous challenge. Among the materials being investigated for target recognition, peptides are attractive candidates because of their chemical robustness, divergence, and their homology to natural olfactory receptors. Using a combinatorial peptide library and either a graphitic surface or phenyl-terminated self-assembled monolayer as relevant target surfaces, we successfully selected three interesting peptides that differentiate a single carbon deviation among benzene and its analogues. The heterogeneity of the designed target surfaces provided peptides with varying affinity toward targeted molecules and generated a set of selective peptides that complemented each other. Microcantilever sensors conjugated with each peptide quantitated benzene, toluene and xylene to sub-ppm levels in real time. The selection of specific receptors for a group of volatile molecules will provide a strong foundation for general approach to individually monitoring VOCs.
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http://dx.doi.org/10.1038/srep09196DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4361882PMC
March 2015

Highly photoluminescent superparamagnetic silica composites for on-site biosensors.

J Mater Chem B 2014 Apr 4;2(14):1938-1944. Epub 2014 Mar 4.

Molecular Recognition Research Center, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791, Korea.

For the development of a quick and effective on-site sensor, a multifunctional composite with concentrating and sensing functions is greatly advantageous. The multifunctional composite with high magnetic susceptibility for concentration and enhanced fluorescence intensity for sensing is synthesized and applied for an on-site sensor. The multifunctional composite is prepared by incorporating a cluster of FeO nanocrystals at the centre and a QD-layer at radial equidistance near the surface of a silica microsphere (diameter ∼0.6 μm). The extinguished fluorescence of the composite, through hybridization with quenching DNAs via aptamers anchored on the composite, resumes instantaneously and quantitatively upon addition of target proteins by displacing the quenching DNAs. The configuration of thrombin binding aptamers and quenching DNAs on the composite is optimized to achieve a quenching efficiency of 94%. Upon simple injection of thrombin solution, binding of thrombin to its specific aptamer on the composite is detected at concentrations as low as 0.35 nM (10 ng mL) within 5 min. The current multifunctional composite system is expected to be a promising platform for on-site biosensors.
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http://dx.doi.org/10.1039/c3tb21331eDOI Listing
April 2014

A new combination MALDI matrix for small molecule analysis: application to imaging mass spectrometry for drugs and metabolites.

Analyst 2012 Dec;137(24):5757-62

Department of Molecular Biotechnology, WCU program, Konkuk University, Seoul, 143-701, Korea.

Since the development of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, this procedure has been specifically used for analyzing proteins or high molecular weight compounds because of the interference of matrix signals in the regions of the low mass range. Recently, scientists have been using a wide range of chemical compounds as matrices that ionize small molecules in a mass spectrometer and overcome the limitations of MALDI mass spectrometry. In this study, we developed a new combination matrix of 3-hydroxycoumarin (3-HC) and 6-aza-2-thiothymine (ATT), which is capable of ionizing small molecules, including drugs and single amino acids. In addition to ionization of small molecules, the combination matrix by itself gives less signals in the low mass region and can be used for performing imaging mass spectrometry (IMS) experiments on tissues, which confirms the vacuum stability of the matrix inside a MALDI chamber. The drug donepezil was mapped in the intact tissue slices of mice simultaneously with a spatial resolution of 150 μm during IMS. IMS analysis clearly showed that intact donepezil was concentrated in the cortical region of the brain at 60 min after oral administration. Our observations and results indicate that the new combination matrix can be used for analyzing small molecules in complex samples using MALDI mass spectrometry.
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http://dx.doi.org/10.1039/c2an35782hDOI Listing
December 2012

Protocol for the use of a silica nanoparticle-enhanced microcantilever sensor-based method to detect HBV at femtomolar concentrations.

Methods Mol Biol 2012 ;903:283-93

Department of Chemical Engineering, Kangwon National University, Kangwon-do, Republic of Korea.

DNA sensors that are capable of detecting specific DNA sequences in a bio-sample have recently been highlighted as a powerful and sensitive approach to detect infectious diseases caused by pathogens such as viruses and bacteria. Generally, DNA samples extracted from biological fluids are amplified by PCR prior to analysis by DNA sensors or directly analyzed by DNA sensors equipped with a signal amplification process. Nanoparticles have recently been used to amplify the sensor signal and have been shown to play an important role in improving the sensitivity of mechanical resonating sensors. This is because the weight of the nanoparticle can increase the change in the resonance response of the mechanical sensor since this signal change is closely related to mass. Here, we introduce an experimental method to detect HBV at femtomolar concentrations using a silica nanoparticle-enhanced microcantilever resonating sensor. This method includes the preparation of detection probe-conjugated silica nanoparticles, immobilization of capture probe on the microcantilever sensor and sandwich type detection of HBV DNA.
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http://dx.doi.org/10.1007/978-1-61779-937-2_19DOI Listing
November 2012
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