Tai Hwan Ha

Oriented Immobilization of Antibodies with GST-Fused Multiple Fc-Specific B-Domains on a Gold Surface

We have constructed a novel platform for the oriented buildup of immunoglobulins on a gold surface for a surface plasmon resonance imaging microarray. To this end, genetically engineered glutathione S-transferase proteins bearing one, two, and three Fc-specific B-domains in protein G from Streptococci (GST-GB1, -GB2, and -GB3, respectively) were produced. In order to tether these GST-GBx proteins specifically, a novel glutathione-derivatized ligand (LA-GSH) was also synthesized from a biaminated tri(ethylene glycol) backbone. Each end of the backbone was further functionalized with a maleimide group for a glutathione modification and a lipoic acid for surface immobilization. The glutathione ligand demonstrated a negligible nonspecific protein adsorption toward other spectator proteins while showing a strong specific association toward GST-GBx proteins. This Fc-specific surface exhibited at least a 2-fold enhancement in the immunoglobulin density (from human and mouse) with its antigen capture capability totally conserved compared to a covalently tethered GBx proteins. A single antibody tethered on the GST-GB3 is estimated to capture two antigens (enhanced green fluorescent protein), and this antigen capture ratio seems to be the most efficient value ever observed.

Structural effects of naphthalimide-based fluorescent sensor for hydrogen sulfide and imaging in live zebrafish.

Hydrogen sulfide (H2S) is an important biological messenger, but few biologically-compatible methods are available for its detection in aqueous solution. Herein, we report a highly water-soluble naphthalimide-based fluorescent probe (L1), which is a highly versatile building unit that absorbs and emits at long wavelengths and is selective for hydrogen sulfide over cysteine, glutathione, and other reactive sulfur, nitrogen, and oxygen species in aqueous solution. We describe turn-on fluorescent probes based on azide group reduction on the fluorogenic ‘naphthalene’ moiety to fluorescent amines and intracellular hydrogen sulfide detection without the use of an organic solvent. L1 and L2 were synthetically modified to functional groups with comparable solubility on the N-imide site, showing a marked change in turn-on fluorescent intensity in response to hydrogen sulfide in both PBS buffer and living cells. The probes were readily employed to assess intracellular hydrogen sulfide level changes by imaging endogenous hydrogen sulfide signal in RAW264.7 cells incubated with L1 and L2. Expanding the use of L1 to complex and heterogeneous biological settings, we successfully visualized hydrogen sulfide detection in the yolk, brain and spinal cord of living zebrafish embryos, thereby providing a powerful approach for live imaging for investigating chemical signaling in complex multicellular systems.

Immobilization of hexa-arginine tagged esterase onto carboxylated gold nanoparticles

Hexa-arginine tagged esterase was efficiently immobilized onto carboxylated gold nanoparticles (AuNP-COOH) and its enzyme activity was investigated by monitoring the absorption spectrum of an enzyme substrate, p-nitrophenol butyrate.

3-Input/1-Output Logic Implementation Demonstrated by DNA Algorithmic Self-Assembly

Although structural DNA nanotechnology is a well-established field, computations performed using DNA algorithmic self-assembly is still in the primitive stages in terms of its adaptability of rule implementation and experimental complexity. Here, we discuss the feasibility of constructing an M-input/ N-output logic gate implemented into simple DNA building blocks. To date, no experimental demonstrations have been reported with M > 2 owing to the difficulty of tile design. To overcome this problem, we introduce a special tile referred to as an operator. We design appropriate binding domains in DNA tiles, and we demonstrate the growth of DNA algorithmic lattices generated by eight different rules from among 256 rules in a 3-input/1-output logic. The DNA lattices show simple, linelike, random, and mixed patterns, which we analyze to obtain errors and sorting factors. The errors vary from 0.8% to 12.8% depending upon the pattern complexity, and sorting factors obtained from the experiment are in good agreement with simulation results within a range of 1-18%.

Growth and Detachment of 5 Helix DNA Ribbons.

We report on the concentration-dependent surface-assisted growth and time-temperature-dependent detachment of one-dimensional 5 helix DNA ribbons (5HR) on a mica substrate. The growth coverage ratio was determined by varying the concentration of the 5HR strands in a test tube, and the detachment rate of 5HR on mica was determined by varying the incubation time at a fixed temperature on a heat block. The topological changes in the concentration-dependent attachment and the time-temperature-dependent detachment for 5HR on mica were observed via atomic force microscopy. The observations indicate that 5HR started to grow on mica at ~10 nM and provided full coverage at ~50 nM. In contrast, 5HR at 65 °C started to detach from mica after 5 min and was completely removed after 10 min. The growth and detachment coverage show a sinusoidal variation in the growth ratio and a linear variation with a rate of detachment of 20%/min, respectively. The physical parameters that control the stability of the DNA structures on a given substrate should be studied to successfully integrate DNA structures for physical and chemical applications.

Fabrication and characterization of PNA–DNA hybrid nanostructures

Although the distinct properties and synthesis methodology of peptide nucleic acid (PNA) molecules have been established by extensive studies, the construction of an artificial nanostructure made from PNA has been examined in only a few reports. Here we study the feasibility of constructing PNA–DNA hybrid nanostructures by conventional free solution annealing and substrate assisted growth methods. For conventional free solution annealing, we introduced a 2-step annealing procedure to mitigate the self-aggregation of PNA in the formation of stable PNA–DNA hybrid structures. Atomic force microscopy images revealed the formation of PNA–DNA hybrid nanostructures smaller than normal DNA and the Raman band intensities of the hybrid gradually decreased as a few DNA strands were replaced by PNA possibly due to the fast binding properties of PNA and the structural stress between PNA and DNA.

Photoreactive immobilization of 11 -(2,4 -dinitro -5 -fluorobenzene )-undecenamide on a hydrogenated silicon (100) surface for protein immobilizations

Several nucleophiles such as proteins or poly(ethyleneimine) could be easily conjugated with a 11-(2,4-dinitro-5-fluorobenzene)undecenamide (DFUA) monolayer photochemically prepared on a silicon (100) surface.