Chanil Chung

PDMS-based micro PCR chip with Parylene coating

We have developed a microchip for polymerase chain reaction (PCR) with polydimethylsiloxane (PDMS). PDMS has good characteristics: it is cheap, transparent, easy to fabricate and biocompatible. But in micro PCR, the porosity of PDMS causes several critical problems such as bubble formation, sample evaporation and protein adsorption. To solve those problems, we coated the micro PCR chips with Parylene film, which has low permeability to moisture and long-term stability. We investigated the influence of low thermal conductivity of PDMS and Parylene on the thermal characteristics of the PCR chips with numerical analysis. The thermal responses of micro PCR chips were compared for three materials: silicon, glass and PDMS. From the results, we identified appropriate thermal responses of the PDMS-based micro PCR chips by heating both the top and bottom sides. We could successfully amplify the angiotensin converting enzyme gene with as small a volume as 2 μl on the PDMS-based micro PCR chips without any additives.

A novel electroporation method using a capillary and wire-type electrode

Electroporation is widely used to achieve gene transfection. A common problem in electroporation is that it has a lower viability than any other transfection method. In this study, we developed a novel electroporation device using a capillary tip and a pipette that was effective on a wide range of mammalian cells, including cell lines, primary cells, and stem cells. The capillary electroporation system considerably reduced cell death during electroporation because of its wire-type electrode, which has a small surface area. The experimental results also indicated that the cell viability was dependent on the change in pH induced by electrolysis during electroporation. Additionally, the use of a long and narrow capillary tube combined with simple pipetting shortened the overall time of the electroporation process by up to 15 min, even under different conditions with 24 samples. These results were supported by comparison with a conventional electroporation system. The transfection rate and the cell viability were enhanced by the use of the capillary system, which had a high transfection rate of more than 80% in general cell lines such as HeLa and COS-7, and more than 50% in hard-to-transfect cells such as stem or primary cells. The viability was approximately 70-80% in all cell types used in this study.

Rapid three-dimensional passive rotation micromixer using the breakup process

Stretching and folding, diffusion, and breakup are three basic processes that occur while mixing fluids. Although stretching and folding the interface of two fluids by rotation enables the mixing at microscale level in both low and high Reynolds number flows, rotation is not as effective at a low Reynolds number as at a high Reynolds number. Therefore, developing a rapid micromixer for microfluidic systems that can be used at a low Reynolds number is a challenging task, because it can demonstrate the full potential of microfluidic systems in commercial markets. Here, to enhance the mixing efficiency of a micromixer based on passive rotation, we present a breakup method. The breakup method not only generates interface actively but also enhances the diffusion process at the interface. With our novel design, over 70% mixing can be achieved only after passing through a 4 mm long microchannel. In this work, the mixer was easily fabricated with polydimethylsiloxane by soft lithography and a self-aligned bonding method with methanol. We analyzed the flow in the micromixer using the computational fluid dynamics method. Also, we conducted quantitative analyses using a confocal scanning microscope and image processing.

Simultaneous counting of two subsets of leukocytes using fluorescent silica nanoparticles in a sheathless microchip flow cytometer

A portable flow cytometer has been recognized as an important tool for many clinical applications such as HIV/AIDS screening in developing countries and regions with limited medical facilities and resources. Conventional flow cytometers typically require multiple detectors for simultaneous identification of multiple subsets of immune cell. To minimize the number of detectors toward portable flow cytometry or to analyze multi-parametric cellular information with minimum number of detectors in conventional flow cytometers, we propose a versatile multiplexed cell-counting method using functional silica nanoparticles (SiNPs). FITC-doped SiNPs, which are 100 times brighter than the FITC molecules itself, were used as new intensity-based fluorescent dye complexes to simultaneously measure two subsets of leukocytes using a single detector. CD45(+)CD4(+) cells tagged with these FITC-doped SiNPs were 50 times brighter than CD45(+)CD4(-) cells tagged only with FITC. To make the overall system compact, a disposable microchip flow cytometer that does not require sheath flow was developed. Combining these dye-doped SiNPs based detection schemes and the sheathless microchip flow cytometer scheme, we successfully identified and counted two subsets of leukocytes simultaneously (R(2) = 0.876). These approaches can be the building blocks for a truly portable and disposable flow cytometer for various clinical cytometry applications.

Serial dilution microchip for cytotoxicity test

Todays pharmaceutical industry is facing challenges resulting from the vast increases in sample numbers produced by high-throughput screening (HTS). In addition, the bottlenecks created by increased demand for cytotoxicity testing (required to assess compound safety) are becoming a serious problem. We have developed a polymer PDMS (polydimethylsiloxane) based microfluidic device that can perform a cytotoxicity test in a rapid and reproducible manner. The concept that the device includes is well adjustable to automated robots in huge HTS systems, so we can think of it as a potential dilution and delivery module. Cytotoxicity testing is all about the dilution and dispensing of a drug sample. Previously, we made a PDMS based microfluidic device which automatically and precisely diluted drugs with a buffer solution with serially increasing concentrations. This time, the serially diluted drug solution was directly delivered to 96 well plates for cytotoxicity testing. Cytotoxic paclitaxel solution with 2% RPMI 1640 has been used while carrying out cancerous cell based cytotoxicity tests. We believe that this rapid and robust use of the PDMS microchip will overcome the growing problem in cytotoxicity testing for HTS.

Functional integration of serial dilution and capillary electrophoresis on a PDMS microchip

For the quantitative analysis of an unknown sample a calibration curve should be obtained, as analytical instruments give relative, rather than absolute measurements. Therefore, researchers should make standard samples with various known concentrations, measure each standard and the unknown sample, and then determine the concentration of the unknown by comparing the measured value to those of the standards. These procedures are tedious and time-consuming. Therefore, we developed a polymer based microfluidic device from polydimethylsiloxane, which integrates serial dilution and capillary electrophoresis functions in a single device. The integrated microchip can provide a one-step analytical tool, and thus replace the complex experimental procedures. Two plastic syringes, one containing a buffer solution and the other a standard solution, were connected to two inlet holes on a microchip, and pushed by a hydrodynamic force. The standard sample is serially diluted to various concentrations through the microfluidic networks. The diluted samples are sequentially introduced through microchannels by electro-osmotic force, and their laser-induced fluorescence signals measured by capillary electrophoresis. We demonstrate the integrated microchip performance by measuring the fluorescence signals of fluorescein at various concentrations. The calibration curve obtained from the electropherograms showed the expected linearity.

Active sealing for soft polymer microchips: method and practical applications

This paper presents a new sealing method for soft polymer (elastomer) microchips. A robust and reversible sealing method, which allows various materials to be bonded and sealed tightly with each other even in aqueous solutions, is developed. A poly (dimethylsiloxane) microchip system, which can actively generate bonding and sealing forces by itself, is invented. By inducing negative pressure into additional closed areas, an instant sucking disc is made. This disc is used to tighten up the conformal contact of soft polymers. Other functionalities of active sealing such as making reusable microchips, patterning cells and performing cellular assays in a single dish have also been examined and will be discussed hereunder. This technique gives a robust and universal solution for microchip sealing issues by sealing soft polymers with diverse materials under various conditions. Active sealing will simplify numerous assays in lab-on-a-chip industry and will open a new era for cellular microchip assays.

Effects of peak anomalies with the hydrophilic or hydrophobic properties of reservoirs during serial injection on a capillary electrophoresis microchip

Several anomalies, e.g., in peak shape, migration time, and baseline drift, all due to pressure-driven backflow, were previously reported to occur during serial injection on capillary electrophoresis (CE) chips. Since these anomalies were worse for polydimethylsiloxane (PDMS) microchips than for glass microchips, reproducible data on PDMS microchips were difficult to obtain. In this paper, we found that these problems were affected by the hydrophilic or hydrophobic properties of the reservoirs on the microchip and demonstrated that these anomalies were reduced by converting the hydrophobic properties of the reservoirs on the PDMS microchip into hydrophilic ones. Thus, compared with hydrophobic reservoirs, hydrophilic reservoirs were suitable for the formation of a stable plug. Several chip designs were suggested to reduce these pressure-driven backflows.

Absolute CD4+ cell count using a plastic microchip and a microscopic cell counter

We have designed and evaluated the performance of a simple, rapid, and affordable method for counting CD4+ T‐cells with the use of plastic microchips. This new system is an adaptation of a “no‐lyse, no‐wash,” volumetric single platform assay, and absolute CD4+ counts are determined with the use of a microscopic scanning cell counter. To assess the CD4+ count test precision and linearity of the system, measured CD4+ counts were compared with two other reference assays (single and dual platform flow cytometry) with the use of 123 clinical samples including samples obtained from 35 HIV‐infected patients, and artificially diluted samples. A correlation between the results from the use of the new method and from the use of the two other reference assays was r = 0.98 for the clinical samples. A dilution test of the new method demonstrated a linearity of r ≥ 0.99, with coefficients of variation ≤7.6% for all concentration levels. Our findings suggest that the new CD4+ counting device can be potentially be applied for other diagnostic procedures that measure quantities of characteristic antigens or other materials on cells.

Comparison of the automated fluorescence microscopic viability test with the conventional and flow cytometry methods

The cell viability test is an essential tool in any laboratory, performing cell‐based studies and clinical laboratory tests. The trypan blue exclusion method is the most popular assay for its simple concept among various diagnostic tools. However, several disadvantages include time‐consuming and labor‐intensive steps with low precision. In this study, we evaluated a new technique for the automatic cell viability measurement with microscopic cell counter and microchip. Upon blood draw from 11 healthy volunteers, Mononuclear cells were separated immediately from the heparinized whole blood, and the viable cells were diluted from 100 to 1%. The cell viability tests were performed simultaneously with following three methods: the conventional manual trypan blue exclusion method; the flow cytometry measurement with propidium iodide stain; and the newly developed microscopic cell counter with microchip. Linearities, precisions, and correlations from three methods were analyzed and compared. The correlations data from the microscopic cell counter were in good agreement with both the conventional trypan blue method (r=0.99, P<0.05) and the flow cytometry (r=0.99, P<0.05), respectively. The precision (2.0–6.2%) and linearity from the microscopic cell counter method with microchip were superior in comparison with the conventional method. The microscopic cell counter with microchip performed well with high precision, linearity, and efficient running time than both the manual trypan blue and the flow cytometry methods. J. Clin. Lab. Anal. 25:90–94, 2011.