Ho Seok Kwak

Fabrication of plasmon length-based surface enhanced Raman scattering for multiplex detection on microfluidic device

The length of bioreceptors plays an important role in signal enhancement of surface-enhanced Raman scattering (SERS) due to amplification of electromagnetic fields generated by the excitation of localized surface plasmons. Herein, intact antibodies (IgG) and Fab fragments conjugated onto gold nanostar were used to fabricate two kinds of immunosensors for measurement of their SERS signals. Using CA125 as the antigen and Rhodamine-6G (R6G)-conjugated immunogolds, a SERS immunosensor was self-assembled by antigen-antibody interaction. The results showed that the SERS signal from the Fab immunosensor was 2.4 times higher than that of the IgG immunosensor. Furthermore, increased hot-spots by silver atom deposition onto the IgG and Fab immunosensor showed 2.1 and 1.4 times higher signals than before enhancement, respectively. For application, based on the Fab immunosensor, a SERS-compatible microfluidic system was designed for multiplex assays to overcome the drawbacks of conventional assays. This system can measure biological specimens directly from bio fluids instead of using a complex microfluidic device containing separation and detection elements. Four approved biomarkers of breast cancer, including cancer antigen (CA125), HER2, epididymis protein (HE4), and Eotaxin-1, were detected from patient-mimicked serum with limits of 15 fM, 17 fM, 21 fM, and 6.5 fM, respectively. The results indicated that the lengths and geometry of the bioreceptors determined the intensity of SERS signal from the interface and cavity of the sandwich immunosensor. Silver atom deposition at the cavity of the immunosensor increased the SERS signal. Finally, the SERS immunosensor built-in microfluidic system improved the performance of multiplex diagnostics.

Microfluidic high-throughput selection of microalgal strains with superior photosynthetic productivity using competitive phototaxis

Microalgae possess great potential as a source of sustainable energy, but the intrinsic inefficiency of photosynthesis is a major challenge to realize this potential. Photosynthetic organisms evolved phototaxis to find optimal light condition for photosynthesis. Here we report a microfluidic screening using competitive phototaxis of the model alga, Chlamydomonas reinhardtii, for rapid isolation of strains with improved photosynthetic efficiencies. We demonstrated strong relationship between phototaxis and photosynthetic efficiency by quantitative analysis of phototactic response at the single-cell level using a microfluidic system. Based on this positive relationship, we enriched the strains with improved photosynthetic efficiency by isolating cells showing fast phototactic responses from a mixture of 10,000 mutants, thereby greatly improving selection efficiency over 8 fold. Among 147 strains isolated after screening, 94.6% showed improved photoautotrophic growth over the parental strain. Two mutants showed much improved performances with up to 1.9- and 8.1-fold increases in photoautotrophic cell growth and lipid production, respectively, a substantial improvement over previous approaches. We identified candidate genes that might be responsible for fast phototactic response and improved photosynthesis, which can be useful target for further strain engineering. Our approach provides a powerful screening tool for rapid improvement of microalgal strains to enhance photosynthetic productivity.

Integrated Microfluidic Platform for Multiple Processes from Microalgal Culture to Lipid Extraction

For economically viable biofuel production from microalgae, it is necessary to develop efficient analytical platforms for quantitative evaluation of different lipid productivities of numerous microalgal species. Currently, microalgal culture, lipid accumulation, and lipid extraction depend on conventional benchtop methods requiring laborious and time-consuming processes. A poly(dimethylsiloxane) (PDMS)-based integrated microfluidic platform was developed to perform multiple steps in sample preparation on a single device for efficient and quantitative analysis of lipid from various microalgal strains. To achieve this goal, a simple microchannel with a micropillar array was integrated to connect the cell chamber and output reservoir, which act as a filtration unit that enables medium change and solvent extraction by fluid injection using a syringe pump. Multiple processes of cell culture, lipid accumulation, and lipid extraction were successfully accomplished using a single device without time-consuming and labor-intensive steps. Various conditions of solvent volume and temperature were investigated to optimize lipid extraction yield in the microfluidic device. The lipid extraction efficiency in the microfluidic system was higher than that in bulk using the same solvent. The lipid extraction efficiency achieved using less toxic aqueous isopropanol on the integrated device was 113.6% of that obtained with the conventional Bligh-Dyer method. Finally, lipid productivities of different microalgal strains grown in the microfluidic device were analyzed and compared. These results demonstrate that this simple integrated microfluidic platform can be applied as an alternative to conventional benchtop methods for efficient sample preparation in microalgal lipid analysis.

Development of an X-Shape airlift photobioreactor for increasing algal biomass and biodiesel production

The aim of this work was to develop a high efficient photobioreactor for increasing biomass and lipid production in microalgae by assessment of the hydrodynamic properties and kLa which are important parameters for improving the algal cultivation efficiency. We designed three different photobioreactors (H-Shape, X-Shape and serial-column). Among them, X-Shape showed the highest hydrodynamic properties and kLa for algal cultivation. Thus, we evaluated the biomass and the lipid production in a 20L scale-up X-Shape photobioreactor. The biomass and lipid production from X-Shape photobioreactor are 1.359±0.007gL-1 and 117.624±3.522mgL-1, respectively; which are 30.05% and 23.49% higher than those from the control photobioreactor. Finally, we observed the lipid from X-Shape had high MUFAs, CN and low IV, which is suitable for high quality of biodiesel, suggesting that it can be practicably utilized for mass production of algal biofuel.

Improvement of Squalene Production from CO2 in Synechococcus elongatus PCC 7942 by Metabolic Engineering and Scalable Production in a Photobioreactor

The push-and-pull strategy for metabolic engineering was successfully demonstrated in Synechococcus elongatus PCC 7942, a model photosynthetic bacterium, to produce squalene from CO2. Squalene synthase (SQS) was fused to either a key enzyme (farnesyl diphosphate synthase) of the methylerythritol phosphate pathway or the β-subunit of phycocyanin (CpcB1). Engineered cyanobacteria with expression of a fusion CpcB1-SQS protein showed a squalene production level (7.16 ± 0.05 mg/L/OD730) that was increased by 1.8-fold compared to that of the control strain expressing SQS alone. To increase squalene production further, the gene dosage for CpcB1·SQS protein expression was increased and the fusion protein was expressed under a strong promoter, yielding 11.98 ± 0.49 mg/L/OD730 of squalene, representing a 3.1-fold increase compared to the control. Subsequently, the best squalene producer was cultivated in a scalable photobioreactor (6 L) with light optimization, which produced 7.08 ± 0.5 mg/L/OD730 squalene (equivalent to 79.2 mg per g dry cell weight). Further optimization for photobioprocessing and strain development will promote the construction of a solar-to-chemical platform.

Quantitative analysis of the chemotaxis of a green alga, Chlamydomonas reinhardtii, to bicarbonate using diffusion-based microfluidic device

There is a growing interest in the photosynthetic carbon fixation by microalgae for the production of valuable products from carbon dioxide (CO2). Microalgae are capable of transporting bicarbonate (HCO3 (-)), the most abundant form of inorganic carbon species in the water, as a source of CO2 for photosynthesis. Despite the importance of HCO3 (-) as the carbon source, little is known about the chemotactic response of microalgae to HCO3 (-). Here, we showed the chemotaxis of a model alga, Chlamydomonas reinhardtii, towards HCO3 (-) using an agarose gel-based microfluidic device with a flow-free and stable chemical gradient during the entire assay period. The device was validated by analyzing the chemotactic responses of C. reinhardtii to the previously known chemoattractants (NH4Cl and CoCl2) and chemotactically neutral molecule (NaCl). We found that C. reinhardtii exhibited the strongest chemotactic response to bicarbonate at the concentration of 26 mM in a microfluidic device. The chemotactic response to bicarbonate showed a circadian rhythm with a peak during the dark period and a valley during the light period. We also observed the changes in the chemotaxis to bicarbonate by an inhibitor of bicarbonate transporters and a mutation in CIA5, a transcriptional regulator of carbon concentrating mechanism, indicating the relationship between chemotaxis to bicarbonate and inorganic carbon metabolism in C. reinhardtii. To the best of our knowledge, this is the first report of the chemotaxis of C. reinhardtii towards HCO3 (-), which contributes to the understanding of the physiological role of the chemotaxis to bicarbonate and its relevance to inorganic carbon utilization.

Rapid selection of astaxanthin-hyperproducing Haematococcus mutant via azide-based colorimetric assay combined with oil-based astaxanthin extraction

The aim of this work was to develop a new approach for simple and high-throughput selection of astaxanthin-hyperproducing Haematococcus mutants through a sequential combination method of azide-based colorimetric assessment and oil-based astaxanthin quantification. Randomly mutagenized cells were spotted on solid culture medium containing 50 µM of sodium azide to accelerate the biosynthesis of astaxanthin. After 3 days, highly-induced mutants were preliminarily isolated by visual inspection and their astaxanthin accumulations were rapidly quantified by soybean oil-based extraction method. On the whole, the selected mutants showed reduced vegetative growth rates but eventually exhibited higher astaxanthin productions than the parental strain owing to their improved inductive growths. Among them, M13 showed 174.7 ± 5.69 mg L-1 of the highest astaxanthin production, which is 1.59-times higher than that of wild-type. This wide-scope screening method expedites both upstream and downstream astaxanthin quantification, making it a useful tool for isolating microalgae with high astaxanthin production.