Nam Su Heo

Solution Chemistry of Self-Assembled Graphene Nanohybrids for High-Performance Flexible Biosensors

We report the preparation of free-standing flexible conductive reduced graphene oxide/Nafion (RGON) hybrid films by a solution chemistry that utilizes self-assembly and directional convective-assembly. The hydrophobic backbone of Nafion provided well-defined integrated structures, on micro- and macroscales, for the construction of hybrid materials through self-assembly, while the hydrophilic sulfonate groups enabled highly stable dispersibility ( approximately 0.5 mg/mL) and long-term stability (2 months) for graphene. The geometrically interlocked morphology of RGON produced a high degree of mechanical integrity in the hybrid films, while the interpenetrating network constructed favorable conduction pathways for charge transport. Importantly, the synergistic electrochemical characteristics of RGON were attributed to high conductivity (1176 S/m), facilitated electron transfer (ET), and low interfacial resistance. Consequently, RGON films obtained the excellent figure of merit as electrochemical biosensing platforms for organophosphate (OP) detection, that is, a sensitivity of 10.7 nA/microM, detection limit of 1.37 x 10(-7) M, and response time of <3 s. In addition, the reliability of RGON biosensors was confirmed by a fatigue test of 100 bending cycles. The strategy described here provides insight into the fabrication of graphene and hybrid nanomaterials from a material perspective, as well as the design of biosensor platforms for practical device applications.

In Vivo Synthesis of Diverse Metal Nanoparticles by Recombinant Escherichia coli

Nanometer-scale metal particles are finding many applications in the fields of biology and nanotechnology owing to their unique optical and magnetic properties. Phytochelatin (PC) and other metal-binding proteins have an ability to bind heavy metals and have been used for heavy-metal removal. Thus, we reasoned that they might be employed for the synthesis of metal nanoparticles (NPs). Herein, we report the in vivo biosynthesis of diverse NPs by recombinant Escherichia coli expressing phytochelatin synthase (PCS) and/or metallothionein (MT). NPs of various metal elements, including semiconducting, alkali-earth, magnetic, and noble metals and rare-earth fluorides, could be synthesized in E. coli. The size of NPs could be tuned on the nanoscale by changing the concentration of metal ions in the medium. Thus, the controlled synthesis of NPs with desirable characteristics for in vitro assays and cellular imaging was possible. Paramagnetic NPs could also be synthesized by using the same system. The strategy of employing recombinant E. coli as an NP factory is generally applicable for the combinatorial synthesis of diverse NPs with a wide range of characteristics. Metal NPs exhibit unique optical, electronic, and magnetic properties, which depend on their composition, size, and structure, and have therefore been explored extensively for various applications in bioand nanotechnology. Several physicochemical processes involving reactions at high temperatures in organic solvents have been employed for the synthesis of these metal NPs. In nature, uniand multicellular organisms are capable of reducing and accumulating metal ions as detoxification and homeostasis mechanisms upon their exposure to metal-ion solutions. For example, CdS quantum crystallites of 2–6 nm in diameter were found to be synthesized intracellularly in Candida glabrata, Schizosaccharomyces pombe, and engineered E. coli. 4] In another example, the fungus Verticillium sp. was able to synthesize gold NPs of 20 nm in diameter by reducing aqueous AuCl4 ions. Also, it is known that 20–30 nm Fe3O4 magnetite nanocrystals can be synthesized by the magnetosome of Magnetospirillum magnetotacticum. 8] These examples demonstrate that microbes can be employed as a factory for metal-NP synthesis. Although the exact mechanisms and identities of associated microbial proteins for metal-NP synthesis are not clear, two cysteine-rich, heavy-metal-binding biomolecules, PC and MT, have been relatively well characterized. PCs are oligoglutathione peptides of varying sizes that are synthesized by PCS and that can form metal complexes with Cd, Cu, Ag, Pb, and Hg, 10] whereas MTs are gene-encoded proteins capable of directly binding Cu, Cd, and Zn. Recently, the in vivo synthesis of CdS nanocrystals by recombinant E. coli expressing the S. pombe PCS gene and the g-glutamylcysteine synthetase gene was reported. However, the versatile capabilities of PC and MT to bind various metal ions and to form NPs have not been fully exploited. Herein, we report a general strategy for the in vivo synthesis of diverse NPs by using recombinant E. coli expressing Arabidopsis thaliana PCS (AtPCS) and/or Pseudomonas putida MT (PpMT). Various metals, including semiconducting (Cd, Se, Zn, Te), alkali-earth (Cs, Sr), magnetic (Fe, Co, Ni, Mn), and noble (Au, Ag) metals and rare-earth fluorides (Pr, Gd), were incubated in assorted combinations with the recombinant E. coli cells for the in vivo synthesis of the corresponding metal NPs. The resulting NPs were analyzed for their optical, magnetic, and physicochemical properties. Recombinant E. coli strains expressing AtPCS, PpMT, or both AtPCS and PpMT were also designed and examined for their ability to synthesize diverse metal NPs. Quantum dots (QDs), fluorescent semiconducting material, were used as an example in experiments to tune the sizes of NPs by varying the concentration of the metal ion in the medium. Finally, functionalized QDs were applied to the in vitro conjugation of biomaterials and cellular-imaging analysis. In vivo NP synthesis in recombinant E. coli is described schematically in Figure S1 of the Supporting Information. [*] Dr. T. J. Park, Prof. S. Y. Lee, N. S. Heo, Prof. T. S. Seo Department of Chemical and Biomolecular Engineering BioProcess Engineering Research Center Center for Systems and Synthetic Biotechnology Institute for the BioCentury, KAIST 335 Gwahangno, Yuseong-gu, Daejeon 305-701 (Republic of Korea) Fax: (+ 82)42-350-8800 E-mail: leesy@kaist.ac.kr Homepage: http://mbel.kaist.ac.kr

Development of label-free optical diagnosis for sensitive detection of influenza virus with genetically engineered fusion protein

An active immobilization method utilizing the metal-binding property was developed and examined for its ability to facilitate the biosensing of avian influenza virus. The special biosensing performance with optical plasmonic analysis, including surface plasmon resonance (SPR) was evaluated on gold substrate and also by SPR imaging (SPRi) and localized SPR (LSPR) system where antigen-antibody interaction occurs. A complete optical analytical system was developed by integrating microarray and fabricating nanoparticles onto a single glass chip, thus allowing specific and sensitive diagnosis with subsequent binding. Reaction condition for the maximum reactivity was optimized by SPR analysis and more sensitive interaction was performed by SPRi analysis. Furthermore, ultra-sensitive detection was successfully developed up to the target molecules of 1 pg mL(-1) by LSPR analysis. The advanced phase-in of enhanced plasmonic sensing system allows more efficient and sensitive detection by switching fabrication processes, which were prepared on the gold surface using the nanoparticles. This inflow contains the gold binding polypeptide (GBP)-fusion protein, which was expressed in recombinant Escherichia coli cells, was bound onto the gold substrates by means of specific interaction. The GBP-fusion method allows immobilization of proteins in bioactive forms onto the gold surface without surface modification suitable for studying antigen-antibody interaction. It was used for the detection of influenza virus, an infectious viral disease, as an example case.

In vitro biosynthesis of metal nanoparticles in microdroplets.

We report the use of a hydrogel polymer, recombinant Escherichia coli cell extracts, and a microdroplet-based microfluidic device to fabricate artificial cellular bioreactors which act as reactors to synthesize diverse metal nanoparticles (NPs). The combination of cell extracts, microdroplet-based microfluidic device, and hydrogel was able to produce a mass amount of artificial cellular bioreactors with uniform size and shape. For the first time, we report the alternating generation of microdroplets through one orifice for the fabrication of the artificial cellular reactors using the cell extract as inner cellular components and hydrogel as an artificial cellular membrane. Notably, the hydrogels were able to protect the encapsulated cell extracts from the surrounding environment and maintain the functionality of cellular component for the further cellular bioreactor applications. Furthermore, the successful applications of the fabricated artificial cellular bioreactors to synthesize various NPs including quantum dots, iron, and gold was demonstrated. By employing this microfluidic technique, the artificial cellular bioreactors could be applicable for the synthesis of diverse metal NPs through simple dipping of the reactors to the metal precursor solutions. Thus, the different size of NPs can be synthesized through controlling the concentration of metal precursors. This artificial cellular bioreactors offer promising abilities to biofriendly ways to synthesis diverse NPs and can be applicable in chemical, biomedical, and bioengineering applications.

Porous Covalent Triazine Polymer as a Potential Nanocargo for Cancer Therapy and Imaging.

A microporous covalent triazine polymer (CTP) network with a high surface area was synthesized via the Friedel-Crafts reaction and employed as a potential transport system for drug delivery and controlled release. The CTP was transformed to the nanoscale region by intense ultrasonication followed by filtration to yield nanoscale CTP (NCTP). This product showed excellent dispersibility in physiological solution while maintaining its chemical structure and porosity. An anticancer drug, doxorubicin (DOX), was loaded onto the NCTP through hydrophobic and π-π interactions, and its release was controlled at pH 4.8 and 7.4. The NCTP showed no toxicity toward cancer or normal cells, but the NCTP-DOX complex showed high efficacy against both types of cells in vitro. In-vitro cell imaging revealed that NCTP is a potential material for bioimaging. The potency of NCTP on cellular senescence was confirmed by the expression of senescence associated marker proteins p53 and p21. These results suggest that NCTP can be used as a new platform for drug delivery and imaging with potential applications in diagnosis and therapy.

CRP detection from serum for chip-based point-of-care testing system

Most of point-of-care testing (POCT) to improve facilitates in diagnosis, treatment, and monitoring of patients. POCT technique has still remained a quantitatively and accurately detective effect. In this article, we demonstrated that real human C-reactive protein (CRP) in serum was detected for a chip-based point-of-care testing application based on a nanogap-embedded field effect transistor (FET), and the results were compared with those obtained via the enzyme-linked immunosorbent assay (ELISA) method. The limit of detection (LOD), determined from the standard curve, was 0.1 ng/ml, which is comparable to that of commercialized ELISAs. We evaluated that an improved detection range (0.1 ng/ml to 100 ng/ml) was achieved by comparing with commercialized ELISA. Control experiments to determine selectivity and to discern false-positive/false-negative rates were also performed. This report is the first description of the detection of CRP in human serum using a silicon-based biosensor.

Development of gold nanoparticle-aptamer-based LSPR sensing chips for the rapid detection of Salmonella typhimurium in pork meat

A non-labeled, portable plasmonic biosensor-based device was developed to enable the ultra-sensitive and selective detection of Salmonella typhimurium in pork meat samples. Specifically, a plasmonic sensor, using the self-assembly of gold nanoparticles (AuNPs) to achieve a regulated diameter of 20 nm for the AuNP monolayers, was used to conduct high-density deposition on a transparent substrate, which produced longitudinal wavelength extinction shifts via a localized surface plasmon resonance (LSPR) signal. The developed aptamers conjugated to the LSPR sensing chips revealed an ultra-sensitive upper limit of detection (LOD) of approximately 104 cfu/mL for S. typhimurium in pure culture under the optimal assay conditions, with a total analysis time of 30–35 min. When the LSPR sensing chips were applied on artificially contaminated pork meat samples, S. typhimurium in the spiked pork meat samples was also detected at an LOD of 1.0 × 104 cfu/mL. The developed method could detect S. typhimurium in spiked pork meat samples without a pre-enrichment step. Additionally, the LSPR sensing chips developed against S. typhimurium were not susceptible to any effect of the food matrix or background contaminant microflora. These findings confirmed that the developed gold nanoparticle-aptamer-based LSPR sensing chips could facilitate sensitive detection of S. typhimurium in food samples.

Label-Free Electrochemical Diagnosis of Viral Antigens with Genetically Engineered Fusion Protein

We have developed a simple electrochemical biosensing strategy for the label-free diagnosis of hepatitis B virus (HBV) on a gold electrode surface. Gold-binding polypeptide (GBP) fused with single-chain antibody (ScFv) against HBV surface antigen (HBsAg), in forms of genetically engineered protein, was utilized. This GBP-ScFv fusion protein can directly bind onto the gold substrate with the strong binding affinity between the GBP and the gold surface, while the recognition site orients toward the sample for target binding at the same time. Furthermore, this one-step immobilization strategy greatly simplifies a fabrication process without any chemical modification as well as maintaining activity of biological recognition elements. This system allows specific immobilization of proteins and sensitive detection of targets, which were verified by surface plasmon resonance analysis and successfully applied to electrochemical cyclic voltammetry and impedance spectroscopy upto 0.14 ng/mL HBsAg.

Microbial inactivation and pesticide removal by remote exposure of atmospheric air plasma in confined environments.

Microbial inactivation and pesticide removal by remote exposure of atmospheric air plasma were investigated in confined environments, including an airtight box and commercial refrigerator. The relative sterilization ratios of remote plasma exposure in an airtight box were found to be affected by the distance from the plasma generator, the volume of box and the time of irradiation; however, over 99% saturation was obtained within only 120 s in all experiments. The sterilization of microorganisms and the removal of pesticide in a refrigerator with a volume of 292 l were also successfully achieved, resulting in over 99% inactivation or decontamination in a few minutes. Considering the reported results by direct plasma exposure and circulation, it can be concluded that the confined environment enhances the efficient irradiation of plasma by eliminating air flow. This system can be applied to the storage to keep agricultural products freshly and exclusion of harmful materials on the products.

Development of a Plastic-Based Microfluidic Immunosensor Chip for Detection of H1N1 Influenza

Lab-on-a-chip can provide convenient and accurate diagnosis tools. In this paper, a plastic-based microfluidic immunosensor chip for the diagnosis of swine flu (H1N1) was developed by immobilizing hemagglutinin antigen on a gold surface using a genetically engineered polypeptide. A fluorescent dye-labeled antibody (Ab) was used for quantifying the concentration of Ab in the immunosensor chip using a fluorescent technique. For increasing the detection efficiency and reducing the errors, three chambers and three microchannels were designed in one microfluidic chip. This protocol could be applied to the diagnosis of other infectious diseases in a microfluidic device.