Keesung Kim

Direct differentiation of human embryonic stem cells into selective neurons on nanoscale ridge/groove pattern arrays

Human embryonic stem cells (hESCs) are pluripotent cells that have the potential to be used for tissue engineering and regenerative medicine. Repairing nerve injury by differentiating hESCs into a neuronal lineage is one important application of hESCs. Biochemical and biological agents are widely used to induce hESC differentiation. However, it would be better if we could induce differentiation of hESCs without such agents because these factors are expensive and it is difficult to control the optimal concentrations for efficient differentiation with reduced side effects. Moreover, the mechanism of differentiation induced by these factors is still not fully understood. In this study, we present evidence that nanoscale ridge/groove pattern arrays alone can effectively and rapidly induce the differentiation of hESCs into a neuronal lineage without the use any differentiation-inducing agents. Using UV-assisted capillary force lithography, we constructed nanoscale ridge/groove pattern arrays with a dimension and alignment that were finely controlled over a large area. Human embryonic stem cells seeded onto the 350-nm ridge/groove pattern arrays differentiated into neuronal lineage after five days, in the absence differentiation-inducing agents. This nanoscale technique could be used for a new neuronal differentiation protocol of hESCs and may also be useful for nanostructured scaffolding for nerve injury repair.

Synergistically enhanced osteogenic differentiation of human mesenchymal stem cells by culture on nanostructured surfaces with induction media.

We have examined the effects of surface nanotopography on in vitro osteogenesis of human mesenchymal stem cells (hMSCs). UV-assisted capillary force lithography was employed to fabricate a scalable (4x5 cm), well-defined nanostructured substrate of a UV curable polyurethane polymer with dots (150, 400, 600 nm diameter) and lines (150, 400, 600 nm width). The influence of osteogenic differentiation of hMSCs was characterized at day 8 by alkaline phosphatase (ALP) assay, RT-PCR, and real-time PCR analysis. We found that hMSCs cultured on the nanostructured surfaces in osteogenic induction media showed significantly higher ALP activity compared to unpatterned PUA surface (control group). In particular, the hMSCs on the 400 nm dot pattern showed the highest level of ALP activity. Further investigation with real-time quantitative RT-PCR analysis demonstrated significantly higher expression of core binding factor 1 (Cbfa1), osteopontin (OP), and osteocalcin (OC) levels in hMSCs cultured on the 400 nm dot pattern in osteogenic induction media. These findings suggest that surface nanotopography can enhance osteogenic differentiation synergistically with biochemical induction substance.

Design of Polydiacetylene-Phospholipid Supramolecules for Enhanced Stability and Sensitivity

We present polydiacetylene (PDA) liposome assemblies with various phospholipids that have different headgroup charges and phase transition temperatures (T(m)). 10,12-Pentacosadiynoic acid (PCDA)-epoxy was used as a base PDA monomer and the insertion of highly charged phospholipids resulted in notable changes in the size of liposome and reduction of the aggregation of PDA liposome. Among the various phospholipids, the phospholipid with a moderate T(m) demonstrated enhanced stability and sensitivity, as measured by the size and zeta potential over storage time, thermochoromic response, and transmission electron microscopy images. By combining these results, we were able to detect immunologically an antibody of bovine viral diarrhea virus over a wide dynamic range of 0.001 to 100 μg/mL.

Biomimetic detection of aminoglycosidic antibiotics using polydiacetylene–phospholipids supramolecules

We rationally designed highly sensitive and selective polydiacetylene (PDA)-phospholipids liposomes for the facile detection of aminoglycosidic antibiotics. The detecting mechanism mimics the cellular membrane interactions between neomycin and phosphatidylinositol-4,5-bisphosphate (PIP(2)) phospholipids. The developed PDA-PIP(2) sensory system showed a detection limit of 61 ppb for neomycin and was very specific to aminoglycosidic antibodies only.

Janus-Compartmental Alginate Microbeads Having Polydiacetylene Liposomes and Magnetic Nanoparticles for Visual Lead(II) Detection

Janus-compartmental alginate microbeads having two divided phases of sensory polydiacetylene (PDA) liposomes and magnetic nanoparticles were fabricated for facile sensory applications. The sensory liposomes are composed of PDA for label-free signal generation and 1,2-dipalmitoyl-sn-glycero-3-galloyl (DPGG) lipids whose galloyl headgroup has specific interactions with lead(II). The second phase having magnetic nanoparticles is designed for convenient handling of the microbeads, such as washing, solvent exchange, stirring, and detection, by applying magnetic field. Selective and convenient colorimetric detection of lead(II) and efficient removal of lead(II) by alginate matrix at the same time are demonstrated.

High-sensitivity detection of oxytetracycline using light scattering agglutination assay with aptasensor.

We present an aptamer‐based biosensor (aptasensor) for rapid and high‐sensitive detection of oxytetracycline (OTC) antibiotic in PBS inside a Y‐channel PDMS microfluidic device. The detection was made by real‐time monitoring of the agglutination assay of ssDNA aptamer‐conjugated polystyrene latex microspheres with proximity optical fibers. The agglutination assay was performed with serially diluted OTC antibiotic solutions using highly carboxylated polystyrene particles of 920 nm diameter conjugated with OTC‐binding ssDNA aptamer. Proximity optical fibers were used to measure the increase in 45° forward light scattering of the aggregated particles by fixing them around the viewing cell of the device with stable angle and distance to the detector. The detection limit was around 100 ppb for the current aptasensor system with the detection time less than 3 min.

Sensitive Mie scattering immunoagglutination assay of porcine reproductive and respiratory syndrome virus (PRRSV) from lung tissue samples in a microfluidic chip

A microfluidic immunosensor utilizing Mie scattering immunoaggultination assay was developed for rapid and sensitive detection of porcine reproductive and respiratory syndrome virus (PRRSV) from lung tissue samples of domesticated pigs. Antibodies against PRRSV were conjugated to the surface of highly carboxylated polystyrene microparticles (diameter=920nm) and mixed with the diluted PRRSV tissue samples in a Y-shaped microchannel. Antibody-antigen binding induced microparticle immunoagglutination, which was detected by measuring the forward 45° light scattering of 380nm incident beam using microcallipered, proximity fiber optics. For comparison, multi-well experiments were also performed using the same optical detection setup. The detection limit was determined to be 10(-3)TCID(50)ml(-1) for PRRSV dissolved in PBS, while those of previous RT-PCR studies for PRRSV were 10(1)TCID(50)ml(-1) (conventional assays) or <1TCID(50)ml(-1) (quantitative real-time assays). Mie scattering simulations were able to predict the shape of the PRRSV standard curve, indicating that any non-linearity of the standard curve can be interpreted purely as an optical phenomenon. Each assay took less than 5min. A strong correlation could be found between RT-PCR and this method for the lung tissue samples, even though their respective detection mechanisms are different fundamentally (nucleic acids for RT-PCR and virus antigens for light scattering immunoagglutination assay). Several different dilution factors were also tested for tissue samples, and 1/10 and 1/100 were found to be usable. If the microfluidic chips are used only once (i.e. without re-using them), both superior sensitivity and satisfactory specificity can be demonstrated. Specificity studies revealed the presence of Type II PRRSV and non-presence of Type I PRRSV and that the microfluidic chip assay could detect Type II North American strain of PRRSV for the animals tested. This work demonstrates the potential of the Mie scattering immunoassay on a microfluidic chip towards real-time detection system for viral pathogens in domesticated animals.

AOT/isooctane reverse micelles with a microaqueous core act as protective shells for enhancing the thermal stability of Chromobacterium viscosum lipase

According to the different environmental systems for lipase reactions, changes in thermal stability were investigated by employing the Chromobacterium viscosum lipase and a two-step series-type deactivation model. The half-life (6.81 h) of the lipase entrapped in reverse micelles at 70 °C was 9.87- and 14.80-fold longer than that in glycerol pool or in aqueous buffer. The deactivation constants for the first and second step (k1 and k2) at all temperatures drastically decreased when the lipase was entrapped in reverse micelles. In particular, k1 (3.84 h(-1)) at 70 °C in reverse micelles was 1.57-fold lower than that in aqueous buffer (6.03 h(-1)). Based on the fluorescence spectrometry, the amount of excited forms of tryptophan and tyrosine increased markedly during the thermal-treatment in aqueous buffer, whereas no significant fluctuation was noted in the reversed micellar system. These results indicated that the encapsulation in reverse micelles could be favorable for preventing the enzyme from heat-induced denaturation.

Rapid detection of Mycoplasma pneumonia in a microfluidic device using immunoagglutination assay and static light scattering

We present real‐time, rapid detection of Mycoplasma pneumonia in PBS inside a Y‐channel PDMS microfluidic device via optical fiber monitoring of latex immunoagglutination. The latex immunoagglutination assay was performed with serially diluted M. pneumonia solutions using highly carboxylated polystyrene particles of 390 and 500 nm diameter conjugated with monoclonal anti‐M. pneumonia. Proximity optical fibers were located around the viewing cell of the device, which were used to measure the increase in 45° forward light scattering of the aggregated particles. The detection limit are slightly less than 50 pg mL−1 both for 390 and 500 nm microspheres and the detection time do not exceed 90 s.

Mussel-Inspired Universal Bioconjugation of Polydiacetylene Liposome for Droplet-Array Biosensors

Most solid-state biosensor platforms require a specific immobilization chemistry and a bioconjugation strategy separately to tether sensory molecules to a substrate and attach specific receptors to the sensory unit, respectively. We developed a mussel-inspired universal conjugation method that enables both surface immobilization and bioconjugation at the same time. By incorporating dopamine or catechol moiety into self-signaling polydiacetylene (PDA) liposomes, we demonstrated efficient immobilization of the PDA liposomes to a wide range of substrates, without any substrate modification. Moreover, receptor molecules having a specificity toward a target molecule can also be attached to the immobilized PDA liposome layer without any chemical modification. We applied our mussel-inspired conjugation method to a droplet-array biosensor by exploiting the hydrophilic nature of PDA liposomes coated on a hydrophobic polytetrafluoroethylene surface and demonstrated selective and sensitive detection of vascular endothelial growth factor down to 10 nM.