Tae k Seo

A Graphene Oxide Based Immuno‐biosensor for Pathogen Detection

The development of novel biosensors for highly sensitive, selective, and rapid pathogen detection is of paramount importance for medical diagnostics, food safety screening, and environmental pollution monitoring. Graphene oxide (GO), which is well known as a promising precursor for graphene, has great potential for use in biosensors because of its unique characteristics such as facile surface modification, high mechanical strength, good water dispersibility, and photoluminescence. Recently, Lu et al., and Liu et al. demonstrated the usefulness of the fluorescence quenching properties of GO in DNA biosensing. Gold nanoparticles (AuNPs) have been shown to be excellent quenchers for organic fluorescent tags, semiconductor nanocrystals, and oxidized carbon nanotubes. Herein, we demonstrate a GO-based immuno-biosensor for detecting a rotavirus as a pathogen model. The detection occurs with high sensitivity and selectivity by GO photoluminescence quenching induced by fluorescence resonance energy transfer (FRET) between GO sheets and AuNPs. A GO-based immuno-biosensor system is shown in Figure 1. GO, which was synthesized by a modified Hummers method, was deposited on an amino-modified glass surface. The disruption of the sp structure of graphene crystals during the oxidation process results in the recombination of electron–hole pairs localized within small sp carbon domains that are embedded in an sp matrix, thus resulting in photoluminescence with a quantum yield of 70.3% relative to the fluorescein dye. A homogenous solution of GO shows a broad fluorescence emission peak around 547 nm upon excitation at 400 nm (Figure S1a,b in the Supporting Information). Since the GO has negatively charged functional groups such as carboxylic acids, hydroxy groups, and epoxides, the GO sheets are bound to the positive surface through an electrostatic interaction, which is strong enough to ensure that the GO sheets are retained during a washing step. The antibodies for rotavirus are immobilized on the GO array by a carbodiimide-assisted amidation reaction, and the rotavirus cell is captured by specific antigen–antibody interaction (Figure 1). The capture of a target cell was verified by observing the fluorescence quenching of GO by FRET between the GO and AuNPs. To realize such a novel GO immuno-biosensor, we first synthesized AuNP-linked antibodies (Ab-DNA-AuNP complexes; Figure S2 in the Supporting Information) which were bridged with 100-mer singlestranded DNA molecules. The DNA molecule was used as a mediator as the synthetic method, which is based on phosphoramidite chemistry, provides facile control of distance between Ab and AuNPs, so the AuNPs are placed close to the GO surface. The high affinity of amino functional groups of the DNA nucleotides for multiple AuNPs leads to an enhancement of the quenching efficiency of GO. When the Ab-DNA-AuNP complexes were selectively bound to the target cells that were attached to the GO arrays, a reduction in the fluorescence emission of GO by quenching was detected, thus enabling the identification of pathogenic target cells. AFM analysis was performed to obtain the topographical profile for each step during the fabrication of GO immunobiosensor (Figure 2). GO sheets ranging from 500 nm to 2 mm in lateral dimension (Figure S1b in the Supporting Information) were dispersed uniformly as monoand bilayers, which were determined by measuring the height (1.1 nm) between the two black marks in Figure 2a. The antibody-linked GO array shows brighter spots, particularly at the edges, and also folded structures, which contain many carboxylic acid groups. The measured height of 11.9 nm corresponds to the theoretical value of an antibody (10–15 nm) and approximately 15.3% area per spot was linked by antibodies. Once the target rotavirus was captured, the height of the complexes increased to approximately 81 nm. Considering that the size Figure 1. Illustration of a GO-based immuno-biosensor.

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

Homogeneous Biogenic Paramagnetic Nanoparticle Synthesis Based on a Microfluidic Droplet Generator

The homeostasis process endows microbes with an intrinsic ability to assemble metal ions to form a metal nanoparticle inside. This nanoparticle synthesis strategy inspired from nature can be adapted by using microorganisms as a nanoparticle factory under mild conditions, such as neutral pH, an aqueous phase, and room temperature. Such an eco-friendly biogenic nanoparticle synthesis has advantages over the conventional chemical methods, which require high temperatures, toxic reagents, and large energy sources. In this sense, Fusarium oxysporum, Escherichia coli, Actinobacter sp., and Staphylococcus aureus were employed for synthesizing Fe3O4, CdS, Fe3S4, and Ag nanoparticles in the bulk state. However, a limitation of the biogenic nanoparticles is the lack of control over the particle size. Heterogeneous nanoparticles are mainly produced, which may be derived from the different reaction conditions, such as the concentration of the metal ions surrounding each cell. 7] As the homogeneity of the nanoparticles, for example paramagnetic nanoparticles, has been demonstrated to be an important factor to be applied for data storage media, ferrofluids, and enhancement agents for magnetic resonance imaging (MRI), the production of the homogeneous biogenic nanoparticles should be accordingly resolved. To overcome polydispersity of biogenic nanoparticles, we used a microfluidics-based droplet generator. The droplet microfluidic systems have been proven as powerful analytical tools by compartmentalizing a number of droplets of femtoliter to microliter volume, by providing uniform and well-defined droplet conditions, and by enabling fast chemical and biological reaction in a high-throughput manner. Chemical synthesis of magnetic iron oxide nanoparticle in the droplets was reported to ameliorate the monodispersity owing to the droplet characteristics. Taking full advantage of droplets, we performed biogenic FeMnmagnetic nanoparticle synthesis with the expectation of an improvement of the size homogeneity by precisely tuning the microenvironmental droplet conditions, including cell numbers, concentration of metal ions, and temperature. We prepared the recombinant E. coli expressing Arabidopsis thaliana phytochelatin synthase (AtPCS) and Pseudomonas putida metallothionein (PpMT). Phytochelatin (PC) and metallothionein (MT) are well known as cysteine-rich and heavy-metal-binding proteins. As PCs, which are enzymatically synthesized peptides, and MTs, which are gene-encoded polypeptides, have demonstrated high metal binding affinity and accumulation capacity inside the cells, we genetically designed and engineered E. coli to express a PC synthase of Arabidopsis thaliana along with an MT of Pseudomonas putida for the synergetic effect of the PpMT-AtPCS fusion protein on assembling metal components for nanoparticle synthesis. A droplet microdevice was designed as shown in Figure 1. There are two aqueous-phase inlets for recombinant E. coli cell solution and metal ion solution, and one oil-phase inlet is for introducing FC 40 and surfactant polyethylene glycol perfluoropolyether (PEG-PFPE). For synthesis of FeMn magnetic nanoparticles, we employed FeSO4·7H2O and MnCl2·4H2O mixture as metal sources, and the flow-focusing structure generated droplets which encapsulate E. coli cells and metal ions. They were efficiently mixed when passing through the serpentine microchannel and stored in the droplet incubation chamber for 5 min.

Generation of conductive PEDOT and graphene composite thin films by a layer-by-layer assembly technique

Poly(3,4-ethylenedioxythiophene) (PEDOT)-based thin film has relatively high conductivity, flexibility and transmittance. However, the improvement for mechanical strength and enhanced conductivity is still required to be adatped for commerical applications. Graphene, a one-atom-thick planar sheet of sp2-bonded carbon atoms is considered as an ideal nanocomposite material for these purposes. In this study, we have developed a PEDOT and graphene composite films, two layered graphene/PEDOT and three layered graphene/PEDOT/graphene, by using a layer-by-layer method.

Graphene oxide-based immunobiosensor for ultrasensitive pathogen detection

In this study, we demonstrated a graphene oxide (GO)-based immunobiosensor system for pathogen detection using fluorescence quenching effect between GO and gold nanoparticles. The fluorescent GO sheets was deposited on an amino-modified glass surface by electrostatic force, and the carboxylate functional group on the GO surface was used to covalently conjugate rotavirus-antibodies to be linked on the surface. The target pathogen, rotavirus, was then incubated and bound by a specific antigen-antibody interaction. Finally, an engineered gold nanoparticle-labeled antibody probe was attached to the captured target cell which complexes enable gold nanoparticles to be close to the GO surface, thereby resulting in the quenching of GO fluorescence signal to identify the pathogen. The more Au NPs lead to drastic fluorescence reduction of GO, allowing a sensitive rotavirus detection, and the maximized GO quenching efficiency is obtained up to 85% at 105 pfu/mL. The unique fluorescence emission property and facile fabrication method of GO sheets from cheap graphite resources provide great potential of GO to be applied for biosensors as well as molecular diagnostics as a novel fluorescence tag.