Namwon Kim

Titer plate formatted continuous flow thermal reactors for high throughput applications: fabrication and testing

A high throughput, multi-well (96) polymerase chain reaction (PCR) platform, based on a continuous flow (CF) mode of operation, was developed. Each CFPCR device was confined to a footprint of 8 × 8 mm2, matching the footprint of a well on a standard micro-titer plate. While several CFPCR devices have been demonstrated, this is the first example of a high-throughput multi-well continuous flow thermal reactor configuration. Verification of the feasibility of the multi-well CFPCR device was carried out at each stage of development from manufacturing to demonstrating sample amplification. The multi-well CFPCR devices were fabricated by micro-replication in polymers, polycarbonate to accommodate the peak temperatures during thermal cycling in this case, using double-sided hot embossing. One side of the substrate contained the thermal reactors and the opposite side was patterned with structures to enhance thermal isolation of the closely packed constant temperature zones. A 99 bp target from a λ-DNA template was successfully amplified in a prototype multi-well CFPCR device with a total reaction time as low as ~5 min at a flow velocity of 3 mm s−1 (15.3 s cycle−1) and a relatively low amplification efficiency compared to a bench-top thermal cycler for a 20-cycle device; reducing the flow velocity to 1 mm s−1 (46.2 s cycle−1) gave a seven-fold improvement in amplification efficiency. Amplification efficiencies increased at all flow velocities for 25-cycle devices with the same configuration.

Surface Modification of Droplet Polymeric Microfluidic Devices for the Stable and Continuous Generation of Aqueous Droplets

Droplet microfluidics performed in poly(methyl methacrylate) (PMMA) microfluidic devices resulted in significant wall wetting by water droplets formed in a liquid-liquid segmented flow when using a hydrophobic carrier fluid such as perfluorotripropylamine (FC-3283). This wall wetting led to water droplets with nonuniform sizes that were often trapped on the wall surfaces, leading to unstable and poorly controlled liquid-liquid segmented flow. To circumvent this problem, we developed a two-step procedure to hydrophobically modify the surfaces of PMMA and other thermoplastic materials commonly used to make microfluidic devices. The surface-modification route involved the introduction of hydroxyl groups by oxygen plasma treatment of the polymer surface followed by a solution-phase reaction with heptadecafluoro-1,1,2,2-tetrahydrodecyl trichlorosilane dissolved in fluorocarbon solvent FC-3283. This procedure was found to be useful for the modification of PMMA and other thermoplastic surfaces, including polycyclic olefin copolymer (COC) and polycarbonate (PC). Angle-resolved X-ray photoelectron spectroscopy indicated that the fluorination of these polymers took place with high surface selectivity. This procedure was used to modify the surface of a PMMA droplet microfluidic device (DMFD) and was shown to be useful in reducing the wetting problem during the generation of aqueous droplets in a perfluorotripropylamine (FC-3283) carrier fluid and could generate stable segmented flows for hours of operation. In the case of PMMA DMFD, oxygen plasma treatment was carried out after the PMMA cover plate was thermally fusion bonded to the PMMA microfluidic chip. Because the appended chemistry to the channel wall created a hydrophobic surface, it will accommodate the use of other carrier fluids that are hydrophobic as well, such as hexadecane or mineral oils.

A microfluidic platform with a free-standing perforated polymer membrane

A membrane architecture that facilitates access from both sides in microfluidic environments provides a flexible platform for the study of biosystems. Here, we report for the first time on a simple and low cost fabrication process via nanoimprint lithography (NIL) for a thin, fully released SU-8 membrane with perforated micro- and sub-micron pores and a modular microfluidic system integrated with the membrane. A modified NIL process which combines thermal and UV NIL was employed to define the pore structures in an SU-8 layer coated on a sacrificial layer. We have demonstrated the production of large area SU-8 membranes of as large as 4 inch diameter that are fully covered with perforated micropores. The released SU-8 membrane was easily integrated as a modular component into a microfluidic system by sandwiching the membrane between two microfluidic chips. Important aspects to reliably produce the membrane architecture such as materials selection and process conditions for fabrication are discussed. After demonstrating selective adsorption of lipid vesicles at the micropore sites of the SU-8 membrane, we have reconstituted lipid bilayers at the micropores within the microfluidic system following the method developed by Suzuki et al (2004 Lab Chip 4 502–5). This implies that the membrane architecture can potentially be used as a microfluidic platform with lipid bilayers that can sustain external mechanical stress for biophysical studies of membrane proteins.

Identification of fluid and substrate chemistry based on automatic pattern recognition of stains

This study proposes that images of stains from 100-nanolitre drops can be automatically identified as signatures of fluid composition and substrate chemistry, for e.g. rapid biological testing. Two datasets of stain images are produced and made available online, one with consumable fluids, and the other with biological fluids. Classification algorithms are used to identify an unknown stain by measuring its similarity to representative examples of predefined categories. The accuracy ranges from 80 to 94%, compared to an accuracy by random assignment of 3 to 4%. Clustering algorithms are also applied to group unknown stain images into a number of clusters each likely to correspond to similar combinations of fluids and substrates. The clustering accuracy ranges from 62 to 80%, compared to an accuracy by random assignment of 3 or 4%. The algorithms were also remarkably accurate at determining the presence or absence of biotin and streptavidin respectively in the liquid and on the glass, the salt composition, or the pH of the solution.

Investigation of Two-Phase Flow in Rectangular Micro-Channels

This study addresses air-water, two-phase flows in micro-channels fabricated on poly-methyl-methacrylate (PMMA) with walls that are partially non-wetting (typical static contact angle 65° in stock form) and not molecularly smooth. Two different types of chips were prepared: Micro-milled micro-channels of aspect ratios 1, 2 and 3 with fixed hydraulic diameter on PMMA and micro-channels of unity aspect ratio replicated using hot embossing of PMMA with a micro-milled brass mold insert. Flow-maps obtained using the same gas-liquid injection geometry and method for the three aspect ratio micro-channels are presented, and regime boundaries are compared with those found by other investigations. The results indicate that the bubbly flow regime boundary is shifted to higher liquid and/or lower gas superficial velocities for the higher aspect ratio channels, while transition to the Annular and Annular-Dry regimes remains the same to within experimental uncertainty. The emphasis of what is presented is on the Segmented flow regime. Regular and irregular Segmented flow regimes of three types are assessed on the basis of the statistical variation in the associated phase length scales from flow observations over a substantial channel length. Comparison between results of the two different injection geometries and micro-channel manufacturing techniques indicate that feedback effects are a significant but not the only cause of segmented flow irregularity. The variability in the size of the liquid plug separating gas bubbles in Segmented flow is found to be substantially higher than that of the bubbles even when the flow is regular (low variability of bubble size). The average bubble length associated with a part of Segmented flows, regular and irregular alike is shown to scale approximately with the capillary number to the 2/3 power (liquid volumetric flow ratio to the −2/3). Irregular Segmented flow is favored by higher liquid superficial velocities, lower liquid volumetric flow ratios and lower channel aspect ratios. Of the three aspect ratios examined, the microchannel with aspect-ratio 3 displayed the broadest window of regular Segmented flow. Two-phase flow pressure drop was measured for test channels of unity aspect ratio. Each flow regime identified on the basis of topological observations is associated with different trends of the pressure drop variation with respect to volumetric flow ratio.© 2008 ASME

Optimization of Geometry for Continuous Flow PCR Devices in a Titer Plate-Based PCR Multi-Reactor Platform

A highly parallel, polymerase chain reaction (PCR) multireactor platform is in high demand to satisfy the high throughput requirements for exploiting the accumulated genetic information from the Human Genome Project. By incorporating continuous flow PCR (CFPCR) devices in a polymer 96-well titer plate format, DNA amplification can be performed with steady-state temperature control and faster reaction speed at lower cost. Prior to the realization of a PCR multi-reactor platform, consisting of a sample delivery chip, a PCR multireactor chip, and a thermal cycler, optimization of the geometry for CFPCR devices in a titer plate-based PCR multi-reactor chip based on manufacturing feasibility is necessary. A prototype PCR multi-reactor chip was designed in a 96-well titer plate format with twelve different CFPCR configurations. High quality metallic, large area mold inserts (LAMIs) were fabricated using an SU-8 based UV-LIGA technique by overplating nickel in SU-8 electroplating templates. Micro molding of polycarbonate (PC) was done using hot embossing, resulting in good replication fidelity over the large surface area. Thermal fusion bonding of the molded PC chips using a custom-made bonding jig yielded acceptable sealing results. The manufacturability investigation throughout the design and the process sequence suggested that the microchannel walls require a minimum width of at least 20 μm and an aspect ratio of 2 for structural rigidity. An optimal CFPCR device for use in a PCR multi-reactor chip can be selected with a series of amplification experiments with the development of a thermal cycler.© 2007 ASME

Fish bone peptide promotes osteogenic differentiation of MC3T3-E1 pre-osteoblasts through upregulation of MAPKs and Smad pathways activated BMP-2 receptor

Fish bone, a by‐product of fishery processing, is composed of protein, calcium, and other minerals. The objective of this study was to investigate the effects of a bioactive peptide isolated from the bone of the marine fish, Johnius belengerii, on the osteoblastic differentiation of MC3T3‐E1 pre‐osteoblasts. Post consecutive purification by liquid chromatography, a potent osteogenic peptide, composed of 3 amino acids, Lys‐Ser‐Ala (KSA, MW: 304.17 Da), was identified. The purified peptide promoted cell proliferation, alkaline phosphatase activity, mineral deposition, and expression levels of phenotypic markers of osteoblastic differentiation in MC3T3‐E1 pre‐osteoblast. The purified peptide induced phosphorylation of mitogen‐activated protein kinases, including p38 mitogen‐activated protein kinase, extracellular regulated kinase, and c‐Jun N‐terminal kinase as well as Smads. As attested by molecular modelling study, the purified peptide interacted with the core interface residues in bone morphogenetic protein receptors with high affinity. Thus, the purified peptide could serve as a potential pharmacological substance for controlling bone metabolism.

Control of Internal Stress for High Quality Nickel Large Area Mold Inserts

Metallic large area mold inserts (LAMIs) are essential for the replication of polymer microfluidic devices. Successful molding of micro- or nanoscale features over large areas is dependent on improving the dimensional control of the mold inserts, particularly those fabricated by electrodeposition using the LIGA or UV-LIGA processes. A systematic approach to controlling the internal stress of the nickel deposits, which was essential for predicting the final flatness of the LAMIs prior to electroplating, was carried out. The internal stress of the nickel deposits from a nickel sulfamate solution was estimated using a bent strip stress measurement method after maintaining electroplating chemicals and conditions and reducing contamination. Over-electroplating of the nickel LAMIs was performed on SU-8 electroplating molds on 150 mm diameter Si wafers. Detailed characterization of the nickel LAMIs to determine the relationship between the overall flatness of the LAMIs and the internal stress identified a suitable process window in terms of the current densities (10–20 mA/cm2 ) and the internal stress (−8.3 ∼ −3.0 MPa) for the high quality nickel LAMIs with an overall flatness of 100 μm.© 2009 ASME

Droplet impinging behavior on surfaces with wettability contrasts

Heterogeneous substrates with moderate and extreme wettability contrasts were fabricated by comprising of superhydrophobic/hydrophilic and superhydrophobic/extremely hydrophilic surfaces, respectively. The interactions of water droplets impinging on the surfaces with sharp wettability contrasts were investigated experimentally. The impinging droplets that slightly touch the hydrophilic or extremely hydrophilic areas on each substrate exhibit a directional rebounding towards the more wetting surfaces, i.e., hydrophilic or extremely hydrophilic surface. The trajectory and landing distance of the rebounded droplets were tailored by controlling the releasing height of the droplet, wetting contrast across the border, and portion of the droplet touching the more wetting surface of the substrates with wettability contrasts. The landing distance of the droplet increases with the increased releasing height and higher wettability contrast across the border. Increasing the portion of the impinging droplet touching the more wetting surface of the heterogeneous substrates leads to the shorter landing distance of rebounded droplets.

Hydrothermal Growth of ZnO Nanowires on UV-Nanoimprinted Polymer Structures

Integration of zinc oxide (ZnO) nanowires on miniaturized polymer structures can broaden its application in multi-functional polymer devices by taking advantages of unique physical properties of ZnO nanowires and recent development of polymer microstructures in analytical systems. In this paper, we demonstrate the hydrothermal growth of ZnO nanowires on polymer microstructures fabricated by UV nanoimprinting lithography (NIL) using a polyurethane acrylate (PUA). Since PUA is a siloxane-urethane-acrylate compound containing the alpha-hydroxyl ketone, UV-cured PUA include carboxyl groups, which inhibit and suppress the nucleation and growth of ZnO nanowires on polymer structures. The presence of carboxyl groups in UV-cured PUA was substantiated by Fourier transform infrared spectroscopy (FTIR), and a Ag thin film was deposited on the nanoimprinted polymer structures to limit their inhibitive influence on the growth of ZnO nanowires. Furthermore, the naturally oxidized Ag layer (Ag2O) reduced crystalline lattice mismatches at the interface between ZnO-Ag during the seed annealing process. The ZnO nanowires grown on the Ag-deposited PUA microstructures were found to have comparable morphological characteristics with ZnO nanowires grown on a Si wafer.