Inseong Hwang

Microfluidic approaches for gene delivery and gene therapy

Recent advances in microfluidics have created new and exciting prospects for gene delivery and therapy. The micro-scaled environment within microfluidic systems enables precise control and optimization of multiple processes and techniques used in gene transfection and the production of gene and drug transporters. Traditional non-viral gene transfection methods, such as electroporation, microinjection and optical gene transfection, are improved from the use of innovative microfluidic systems. Additionally, microfluidic systems have also made the production of many viral and non-viral vectors controlled, automated, and reproducible. In summary, the development and application of microfluidic systems are producing increased efficiency in gene delivery and promise improved gene therapy results.

Computational design of protein therapeutics

Computation is increasingly used to guide protein therapeutic designs. Some of the potential applications for computational, structure-based protein design include antibody affinity maturation, modulation of protein-protein interaction, stability improvement and minimization of protein aggregation. The versatility of a computational approach is that different biophysical properties can be analyzed on a common framework. Developing a coherent strategy to address various protein engineering objectives will promote synergy and exploration. Advances in computational structural analysis will thus have a transformative impact on how protein therapeutics are engineered in the future.:

Fabrication and characterization of plasma-polymerized poly(ethylene glycol) film with superior biocompatibility.

A newly fabricated plasma-polymerized poly(ethylene glycol) (PP-PEG) film shows extremely low toxicity, low fouling, good durability, and chemical similarity to typical PEG polymers, enabling live cell patterning as well as various bioapplications using bioincompatible materials. The PP-PEG film can be overlaid on any materials via the capacitively coupled plasma chemical vapor deposition (CCP-CVD) method using nontoxic PEG200 as a precursor. The biocompatibility of the PP-PEG-coated surface is confirmed by whole blood flow experiments where no thrombi and less serum protein adsorption are observed when compared with bare glass, polyethylene (PE), and polyethylene terephthalate (PET) surfaces. Furthermore, unlike bare PE films, less fibrosis and inflammation are observed when the PP-PEG-coated PE film is implanted into subcutaneous pockets of mice groin areas. The cell-repellent property of PP-PEG is also verified via patterning of mammalian cells, such as fibroblasts and hippocampal neurons. These results show that our PP-PEG film, generated by the CCP-CVD method, is a biocompatible material that can be considered for broad applications in biomedical and functional materials fields.

Investigation of sulfhydryl groups in cabbage phospholipase D by combination of derivatization methods and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

All eight cysteine residues in 92 kDa cabbage phospholipase D (PLD), deduced from the cDNA sequence, were shown to have free sulfhydryl groups by analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) of tryptic peptides of PLD derivatized with p-chloromercurybenzoate, iodoacetic acid, and N-ethylmaleimide, as well as of underivatized PLD. Assignment of sulfhydryl groups by any one method was not conclusive. However, complementary information derived from tryptic peptides derivatized with different reagents made full assignment of sulfhydryl groups possible.

Biotin-assisted folding of streptavidin on the yeast surface

Yeast surface display allows heterologously expressed proteins to be targeted to the exterior of the cell wall and thus has a potential as a biotechnology platform. In this study, we report the successful display of functional streptavidin on the yeast surface. Streptavidin binds the small molecule biotin with high affinity (Kd ∼10−14 M) and is used widely in applications that require stable noncovalent interaction, including immobilization of biotinylated compounds on a solid surface. As such, engineering functional streptavidin on the yeast surface may find novel uses in future biotechnology applications. Although the molecule does not require any post‐translational modification, streptavidin is difficult to fold in bacteria. We show that Saccharomyces cerevisiae can fold the protein correctly if induced at 20°C. Contrary to a previous report, coexpression of anchored and soluble streptavidin subunits is not necessary, as expressing the anchored subunit alone is sufficient to form a functional complex. For unstable monomer mutants, however, addition of free biotin during protein induction is necessary to display a functional molecule, suggesting that biotin helps the monomer fold. To show that surface displayed streptavidin can be used to immobilize other biomolecules, we used it to capture biotinylated antibody, which is then used to immunoprecipitate a protein target.

Selective and Direct Immobilization of Cysteinyl Biomolecules by Electrochemical Cleavage of Azo Linkage

Controlled orientation and reserved activity of biomolecules, when site-selectively immobilized in a highly integrated manner on a minimal time scale, are crucial in designing biosensors for the multiplex detection. Here, we describe a novel method for the orientation-controlled immobilization of biomolecules based on site-selective electrochemical activation of p-hydroxyazobenzene self-assembled monolayer (SAM) followed by one-step coupling of cysteinyl biomolecules. The p-aminophenol, a product of reductive cleavage of p-hydroxyazobenzene, was subsequently oxidized to yield p-quinoneimine which then conjugated with cysteinyl biomolecules through 1,4-Michael addition, thus obviating additional linker agents and the related time consumption. Using this method, we selectively activated the electrode surface and immobilized laminin peptide IKVAV, a neurite promoting motif. When we cultured hippocampal neurons on the electrode, the extended neurites were found only within the electrochemically activated area. Hence, the proposed method represents a new promising platform for the patterning of functional peptides, active proteins, and live cells.

Multiplex Immunoassays using Virus-Tethered Gold Microspheres by DC Impedance-based Flow Cytometry

Bead-based multiplex immunoassays for common use require enhanced sensitivity and effective prevention of non-specific adsorption, as well as miniaturization of the detection device. In this work, we have implemented virus-tethered gold microspheres for multiplex immunoassay applications, employing a DC impedance-based flow cytometer as a detection element. The advantages of virus-tethered gold microspheres, including excellent prevention of non-specific adsorption, are extended to signal enhancement arising from the large quantity of antibody loading on each virion, and to flexible movement of filamentous virus. Individual virus-tethered beads generate their own DC impedance and fluorescence signals, which are simultaneously detected by a chip-based microfluidic flow cytometer. This system successfully realized multiplex immunoassays involving four biomarkers: cardiac troponin I (cTnI), prostate specific antigen (PSA), creatine kinase MB (CK-MB), and myoglobin in undiluted human sera, elevating sensitivity by up to 5.7-fold compared to the beads without virus. Constructive integration between filamentous virus-tethered Au-layered microspheres and use of a microfluidic cytometer suggests a promising strategy for competitive multiplex immunoassay development based on suspension arrays.

Robust Type-specific Hemisynapses Induced by Artificial Dendrites.

Type-specificity of synapses, excitatory and inhibitory, regulates information process in neural networks via chemical neurotransmitters. To lay a foundation of synapse-based neural interfaces, artificial dendrites are generated by covering abiotic substrata with ectodomains of type-specific synaptogenic proteins that are C-terminally tagged with biotinylated fluorescent proteins. The excitatory artificial synapses displaying engineered ectodomains of postsynaptic neuroligin-1 (NL1) induce the formation of excitatory presynapses with mixed culture of neurons in various developmental stages, while the inhibitory artificial dendrites displaying engineered NL2 and Slitrk3 induce inhibitory presynapses only with mature neurons. By contrast, if the artificial dendrites are applied to the axonal components of micropatterned neurons, correctly-matched synaptic specificity emerges regardless of the neuronal developmental stages. The hemisynapses retain their initially established type-specificity during neuronal development and maintain their synaptic strength provided live neurons, implying the possibility of durable synapse-based biointerfaces.

Translocation Pathway‐Dependent Assembly of Streptavidin‐ and Antibody‐Binding Filamentous Virus‐Like Particles

Compared to well-tolerated p3 fusion, the display of fast-folding proteins fused to the minor capsid p7 and the major capsid p8, as well as in vivo biotinylation of biotin acceptor peptide (AP) fused to p7, are found to be markedly inefficient using the filamentous phage. Here, to overcome such limitations, the effect of translocation pathways, amber mutation, and phage and phagemid display systems on p7 and p8 display of antibody-binding domains are examined, while comparing the level of in vivo biotinylation of AP fused to p7 or p3. Interestingly, the in vivo biotinylation of AP occurs only in p3 fusion and the fast-folding antibody-binding scaffolds fused to p7 and p8 are best displayed via a twin-arginine translocation pathway in TG1 cells. The lower the expression level of the wild-type p8 and the smaller the size of the guest protein, the better the display of Z-domain fused to the recombinant p8. The in vivo biotinylated multifunctional filamentous virus-like particles can be vertically immobilized on streptavidin (SAV)-coated microspheres to resemble cellular microvilli-like structures, which reportedly enhance protein-protein interactions due to dramatically expanded flexible surface area.

Chemically Deposited Cobalt-Based Oxygen-Evolution Electrocatalysts on DOPA-Displaying Viruses

Filamentous bacteriophages were engineered to display 3,4‐dihydroxy‐l‐phenylalanine (DOPA) onto which a cobalt oxide based oxygen‐evolution catalyst (Co‐OEC) spontaneously deposited at room temperature. Relative to the electrodeposited Co‐OEC, the catalyst formed on the virus exhibited improved durability and current density in a small overpotential region, as well as decreased oxidation states for the formation of Co(OH)2, as confirmed by electrochemical and X‐ray photoelectron spectroscopy analysis. Chemically deposited Co(OH)2 on the virus was oxidized to CoOOH during the water‐oxidation process. Thus, the DOPA‐displaying virus illustrates the affordability of a genetically engineered bacteriophage as a molecular support that can improve the catalytic performance of inorganic materials without any additional electrical energy.