Hyun Soo Kim

Whole-Cell Impedance Analysis for Highly and Poorly Metastatic Cancer Cells

A micro electrical impedance spectroscopy (muEIS) system has been developed and implemented to analyze highly and poorly metastatic head and neck cancer (HNC) cell lines with single-cell resolution. The microsystem has arrays of 16 impedance analysis sites, each of which is capable of capturing a single cell and analyzing its whole-cell electrical impedance spectrum. This muEIS system was used to obtain the electrical impedance spectra of the poorly metastatic HNC cell line 686 LN and the highly metastatic HNC cell line 686 LN-M4e over a frequency range of 40 Hz - 10 MHz. The 686 LN cells had higher impedance phase compared to that of 686 LN-M4e cells at frequencies between 50 kHz and 2 MHz. This result demonstrates that the metastatic state of HNC cells can be distinguished using the developed muEIS system. This system is expected to serve as a powerful tool for future detection and quantification of cancer cells from various tumor stages.

A microfluidic photobioreactor array demonstrating high-throughput screening for microalgal oil production†

Microalgae are envisioned as a future source of renewable oil. The feasibility of producing high-value biomolecules from microalgae is strongly dependent on developing strains with increased productivity and environmental tolerance, understanding algal gene regulation, and optimizing growth conditions for higher production of target molecules. We present a high-throughput microfluidic microalgal photobioreactor array capable of applying 64 different light conditions to arrays of microscale algal photobioreactors and apply this device to investigate how light conditions influence algal growth and oil production. Using the green colony-forming microalga Botryococcus braunii, the light intensity and light-dark cycle conditions were identified that induced 1.8-fold higher oil accumulation over the typically used culture conditions. Additionally, the studies revealed that the condition under which maximum oil production occurs is significantly different from that of maximum growth. This screening test was accomplished using the developed photobioreactor array at 250 times higher throughput compared to conventional flask-scale photobioreactors.

Microfabricated devices in microbial bioenergy sciences.

Microbes provide a platform for the synthesis of clean energy from renewable resources. Significant investments in discovering new microbial systems and capabilities, discerning the molecular mechanisms that mediate microbial bioenergy production, and optimizing existing microbial bioenergy systems have been made. However, further development is needed to achieve the economically feasible large-scale production of value-added energy products. Microfabricated lab-on-a-chip systems provide cost- and time-efficient opportunities for analyzing microbe-mediated bioenergy synthesis. Here, we review developments in the application of lab-on-a-chip systems to the bioenergy sciences. We focus on systems that support the analysis of microbial generation of bioelectricity, biogas, and liquid transportation fuels. We conclude by suggesting possible future directions.

Development of a real-time microchip PCR system for portable plant disease diagnosis.

Rapid and accurate detection of plant pathogens in the field is crucial to prevent the proliferation of infected crops. Polymerase chain reaction (PCR) process is the most reliable and accepted method for plant pathogen diagnosis, however current conventional PCR machines are not portable and require additional post-processing steps to detect the amplified DNA (amplicon) of pathogens. Real-time PCR can directly quantify the amplicon during the DNA amplification without the need for post processing, thus more suitable for field operations, however still takes time and require large instruments that are costly and not portable. Microchip PCR systems have emerged in the past decade to miniaturize conventional PCR systems and to reduce operation time and cost. Real-time microchip PCR systems have also emerged, but unfortunately all reported portable real-time microchip PCR systems require various auxiliary instruments. Here we present a stand-alone real-time microchip PCR system composed of a PCR reaction chamber microchip with integrated thin-film heater, a compact fluorescence detector to detect amplified DNA, a microcontroller to control the entire thermocycling operation with data acquisition capability, and a battery. The entire system is 25×16×8 cm3 in size and 843 g in weight. The disposable microchip requires only 8-µl sample volume and a single PCR run consumes 110 mAh of power. A DNA extraction protocol, notably without the use of liquid nitrogen, chemicals, and other large lab equipment, was developed for field operations. The developed real-time microchip PCR system and the DNA extraction protocol were used to successfully detect six different fungal and bacterial plant pathogens with 100% success rate to a detection limit of 5 ng/8 µl sample.

Micropatterning of poly(dimethylsiloxane) using a photoresist lift-off technique for selective electrical insulation of microelectrode arrays.

A poly(dimethylsiloxane) (PDMS) patterning method based on a photoresist lift-off technique to make an electrical insulation layer with selective openings is presented. The method enables creating PDMS patterns with small features and various thicknesses without any limitation in the designs and without the need for complicated processes or expensive equipments. Patterned PDMS layers were created by spin-coating liquid phase PDMS on top of a substrate having sacrificial photoresist patterns, followed by a photoresist lift-off process. The thickness of the patterned PDMS layers could be accurately controlled (6.5-24 µm) by adjusting processing parameters such as PDMS spin-coating speeds, PDMS dilution ratios, and sacrificial photoresist thicknesses. PDMS features as small as 15 µm were successfully patterned and the effects of each processing parameter on the final patterns were investigated. Electrical resistance tests between adjacent electrodes with and without the insulation layer showed that the patterned PDMS layer functions properly as an electrical insulation layer. Biocompatibility of the patterned PDMS layer was confirmed by culturing primary neuron cells on top of the layer for up to two weeks. An extensive neuronal network was successfully formed, showing that this PDMS patterning method can be applied to various biosensing microdevices. The utility of this fabrication method was further demonstrated by successfully creating a patterned electrical insulation layer on flexible substrates containing multi-electrode arrays.

A droplet microfluidics platform for rapid microalgal growth and oil production analysis.

Microalgae have emerged as a promising source for producing future renewable biofuels. Developing better microalgal strains with faster growth and higher oil production rates is one of the major routes towards economically viable microalgal biofuel production. In this work, we present a droplet microfluidics‐based microalgae analysis platform capable of measuring growth and oil content of various microalgal strains with single‐cell resolution in a high‐throughput manner. The platform allows for encapsulating a single microalgal cell into a water‐in‐oil emulsion droplet and tracking the growth and division of the encapsulated cell over time, followed by on‐chip oil quantification. The key feature of the developed platform is its capability to fluorescently stain microalgae within microdroplets for oil content quantification. The performance of the developed platform was characterized using the unicellular microalga Chlamydomonas reinhardtii and the colonial microalga Botryococcus braunii. The application of the platform in quantifying growth and oil accumulation was successfully confirmed using C. reinhardtii under different culture conditions, namely nitrogen‐replete and nitrogen‐limited conditions. These results demonstrate the capability of this platform as a rapid screening tool that can be applied to a wide range of microalgal strains for analyzing growth and oil accumulation characteristics relevant to biofuel strain selection and development. Biotechnol. Bioeng. 2016;113: 1691–1701.

In-droplet cell concentration using dielectrophoresis

Concentrating cells or adjusting the concentration of cells are one of the most fundamental steps in cell biology experiments, and are typically achieved through centrifugation. However this step is challenging to implement in droplet microfluidics. Here we present an in-droplet cell concentrator that operates by first gradually focusing cells inside a droplet to one side of the droplet using negative dielectrophoresis (nDEP), followed by asymmetric droplet splitting using a Y-shaped junction, resulting in two daughter droplets, one of which containing all or most of the cells. The developed platform was first characterized using droplets containing different number of polystyrene (PS) particles and by varying the applied voltages, flow rates, and the width ratios of the droplet splitting microchannels. Using this platform, the volume of one daughter droplet could be reduced up to 84% compared to that of the mother droplet, which resulted in the PS particle concentration to increase by 5.6-fold, with an average recovery rate of 90%. When testing with cells (Chlamydomonas reinhardtii), recovery rates as high as 98% could be achieved while increasing the cell concentration by 5-fold. This technology adds a new capability to droplet microfluidics operation, and can be used for adjusting concentrations of cells in droplets, exchanging solutions in which cells are suspended in droplets (including cell washing steps), and separating cells of different dielectric properties inside droplets, all of which are common steps in conventional cell assays but have been so far difficult to achieve in droplet format.

A large-scale on-chip droplet incubation chamber enables equal microbial culture time

In many droplet-based screening applications, applying an equal incubation or reaction time to a large number of droplets is critical since differences in incubation time can lead to false positive or false negative hits during the selection process. Even though many droplet incubation platforms have been successfully utilized, having all droplets incubated for equal duration remains a formidable challenge. Here, we present an on-chip droplet incubation chamber that is capable of providing a constant droplet incubation time for a large number of droplets. This first-in first-out droplet incubation chamber essentially functions as a delay compartment to ensure that all droplets have equal incubation time inside the chamber before moving on to the post-incubation assay steps. The functionality of the developed chamber was tested by tracking color dye droplets flowing through the on-chip incubation chamber as well as comparing the growth of droplet-encapsulated cells of a filamentous fungus Fusarium verticillioides. The incubation time can be easily adjusted by changing the volume of the chamber or droplet collection speed. The chamber can also be easily integrated on-chip with other droplet microfluidics functional modules such as droplet generators, detectors, and sorters without any valve and tubing connection.

A Microchip for High-Throughput Axon Growth Drug Screening

It has been recently known that not only the presence of inhibitory molecules associated with myelin but also the reduced growth capability of the axons limit mature central nervous system (CNS) axonal regeneration after injury. Conventional axon growth studies are typically conducted using multi-well cell culture plates that are very difficult to use for investigating localized effects of drugs and limited to low throughput. Unfortunately, there is currently no other in vitro tool that allows investigating localized axonal responses to biomolecules in high-throughput for screening potential drugs that might promote axonal growth. We have developed a compartmentalized neuron culture platform enabling localized biomolecular treatments in parallel to axons that are physically and fluidically isolated from their neuronal somata. The 24 axon compartments in the developed platform are designed to perform four sets of six different localized biomolecular treatments simultaneously on a single device. In addition, the novel microfluidic configuration allows culture medium of 24 axon compartments to be replenished altogether by a single aspiration process, making high-throughput drug screening a reality.

High-throughput droplet microfluidics screening platform for selecting fast-growing and high lipid-producing microalgae from a mutant library

Abstract Biofuels derived from microalgal lipids have demonstrated a promising potential as future renewable bioenergy. However, the production costs for microalgae‐based biofuels are not economically competitive, and one strategy to overcome this limitation is to develop better‐performing microalgal strains that have faster growth and higher lipid content through genetic screening and metabolic engineering. In this work, we present a high‐throughput droplet microfluidics‐based screening platform capable of analyzing growth and lipid content in populations derived from single cells of a randomly mutated microalgal library to identify and sort variants that exhibit the desired traits such as higher growth rate and increased lipid content. By encapsulating single cells into water‐in‐oil emulsion droplets, each variant was separately cultured inside an individual droplet that functioned as an independent bioreactor. In conjunction with an on‐chip fluorescent lipid staining process within droplets, microalgal growth and lipid content were characterized by measuring chlorophyll and BODIPY fluorescence intensities through an integrated optical detection system in a flow‐through manner. Droplets containing cells with higher growth and lipid content were selectively retrieved and further analyzed off‐chip. The growth and lipid content screening capabilities of the developed platform were successfully demonstrated by first carrying out proof‐of‐concept screening using known Chlamydomonas reinhardtii mutants. The platform was then utilized to screen an ethyl methanesulfonate (EMS)‐mutated C. reinhardtii population, where eight potential mutants showing faster growth and higher lipid content were selected from 200,000 examined samples, demonstrating the capability of the platform as a high‐throughput screening tool for microalgal biofuel development.