Wang-Chou Sung

Active micro-mixers using surface acoustic waves on Y-cut 128° LiNbO3

This study presents an active method for micro-mixers using surface acoustic waves (SAW) to rapidly mix co-fluent fluids. Mixing is challenging work in microfluidic systems due to their low-Reynolds-number flow conditions. SAW devices were fabricated on 128? Y-cut lithium niobate (LiNbO3). The micro-mixers are these piezoelectric actuators integrated with polydimethylsiloxane microchannels. The effects of the applied voltages on interdigitated transducers (IDTs) and two layouts, parallel- and transversal-type, of micro-mixers on the mixing performance were experimentally explored. The experimental results revealed that the parallel-type mixer achieved a higher mixing effect. Meanwhile, a higher applied voltage on the IDTs led to a significant improvement in the mixing performance of the active micro-mixer. Typical temperature effects associated with the applied voltages on the IDTs were also investigated. Finally, a digestion reaction between a protein (hemoglobin) and an enzyme (trypsin) was performed to verify the capability of the micro-mixers. The protein?enzyme mixture was qualitatively analyzed using mass spectrometry. Using these SAW-based mixers, the amount of digested peptides increased. Additionally, the protein?enzyme mixture was also quantitatively analyzed using high-performance liquid chromatography. Experimental data showed that the amount of digested peptides increased 21.1% using the active mixer. Therefore, the developed micro-mixers can be applied in microfluidic systems for improving mixing efficiency and thus enhancing the bio-reaction.

A disposable poly(methylmethacrylate)-based microfluidic module for protein identification by nanoelectrospray ionization-tandem mass spectrometry.

The design, fabrication, and analytical use of a poly(methylmethacrylate) (PMMA)‐based microfluidic module for nanoelectrospray ionization‐tandem mass spectrometry (nano‐ESI‐MS/MS) were described. The microfluidic module can be mass‐produced at low costs and used as a disposable device to generate nano‐ESI‐MS/MS signals for protein identification from low amounts of protein samples. Compared with commercially available nanospray capillary tips, the module gave comparable signal quality and also offered advantages in convenience and easiness of operation, permitting repeated usage, and disposability.

Plastic microchip electrophoresis for genetic screening: The analysis of polymerase chain reactions products of fragile X (CGG)n alleles

Clinical screening of abnormal chromosomes associated with fragile X syndrome (FXS) demands a high‐throughput method including DNA sizing and detection of the amplified products. This study is to explore the use of polymer microchip electrophoresis for the analysis of polymerase chain reaction (PCR) products of fragile X (CGG)n alleles to facilitate a fast exclusion test of FXS. The sequences flanking the CGG‐repeat of FMR1 gene was amplified by betaine‐PCR and the amplified products were desalted and then analyzed by microchips which were fabricated on poly(methyl methacrylate) (PMMA) substrate. The PCR bands with more than six CGG‐repeats in difference could be clearly distinguished in less than 3 min by microchip electrophoresis with a separation lenght of 6 cm. It was found that the signal was greatly enhanced with the use of both covalent (Cy5) and intercalating dye (TORRO‐3), which has never been demonstrated before. We tested the method by reanalysis of twelve samples from males and six samples from females. For female samples with less than six repeat differences, Southern blotting method was performed to confirm or exclude the findings from microchips. It was found that the test results from all male and female samples show a 100% correlation between the microchip electrophoresis and the existing methods.

Recent advances in pharmacokinetic applications of capillary electrophoresis.

This article reviews recent capillary electrophoresis (CE)‐based assays which were published for pharmacokinetic studies. Both the advantages and disadvantages of these CE‐based assays are discussed based on their feasibility and the significance towards the better understanding of pharmacokinetics. In addition, as a future outlook, novel assays such as immunoaffinity CE and chip‐based CE for analyzing drugs in biological fluids are summarized in view of their potential for pharmacokinetic applications.

The role of a nuclear protein, histone H1, on signalling pathways for the maturation of dendritic cells.

We have demonstrated previously that liver allograft tolerance is associated with the immunosuppressive activity of anti‐histone H1 autoreactive antibodies induced in the serum of liver transplantation. Furthermore, we and others have shown that nuclear proteins such as histone H1 and high mobility group box 1 play an important role in maturation of dendritic cells (DCs), although the precise mechanisms are still unknown. In the present study, we focus upon the significance of histone H1 on DCs in terms of the intracellular signalling pathway of DCs. Our immunostaining and immunoblot studies demonstrated that histone H1 was detected in cytoplasm and culture supernatants upon the activation of DCs. Histone H1 blockage by anti‐histone H1 antibody down‐regulated the intracellular activation of mitogen‐activated protein kinases (MAPKs) (p38) and IκBα of DCs, and inhibited DC activity in the proliferation of CD4+ T cells. On the other hand, the addition of histone H1 without endotoxin stimulation up‐regulated major histocompatibility complex class II, the CD80 and CD86 surface markers of DCs and the activation of MAPKs (p38 and extracellular‐regulated kinase 1/2) and IκBα. These results suggest that the translocation of histone H1 from nuclei to cytoplasm and the release of their own histone H1 are necessary for the maturation of DCs and the activation for T lymphocytes.

Microfabricated plastic chips by hot embossing methods and their applications for DNA separation and detection

Design and fabrication of microfluidic devices on polymethylmethacrylate (PMMA) substrates using novel microfabrication methods are described. The image of microfluidic devices is transferred from quartz master templates possessing inverse image of the devices to plastic plates by using hot embossing method. The micro channels on master templates are formed by the combination of metal etch mask and wet chemical etching. The micromachined quartz templates can be used repeatedly to fabricate cheap and disposable plastic devices. The reproducibility of the hot embossing method is evaluated after using 10 channels on different plastics. The relative standard deviation of the plastic channel profile from ones on quartz templates is less than 1%. In this study, the PMMA chips have been demonstrated as a micro capillary electrophoresis ((mu) -CE) device for DNA separation and detection. The capability of the fabricated chip for electrophoretic injection and separation is characterized via the analysis of DNA fragments (phi) X174. Results indicate that all of the 11 DNA fragments of the size marker could be identified in less than 3 minutes with relative standard deviations less than 0.4% and 8% for migration time and peak area, respectively. Moreover, with the use of near IR dye, fluorescence signals of the higher molecular weight fragments (

Plastic Microchip Electrophoresis for Clinical Applications of DNA Analysis

GTR 603 bp in length) could be detected at total DNA concentrations as low as 0.1 (mu) g/mL. In addition to DNA fragments (phi) X174, DNA sizing of hepatitis C viral (HCV) amplicon is also achieved using microchip electrophoresis fabricated on PMMA substrate.

Microfluidic Device with Integrated Protein Digestion, Peptide Separation and Nanospray Interface on Poly (Dimethylsiloxane) PDMS Substrate

Microfluidic devices were fabricated on poly(methyl methacrylate) (PMMA) substrate using heat embossing method. Results indicated that all of the 11 DNA fragments of the size marker (HaeIII digest of φX174) could be identified in less than 3 minutes with relative standard deviations less than 0.4% and 8% for migration time and peak area, respectively. The characterized microchip was investigated for clinical post-PCR analysis of hepatitis C virus (HCV) using Laser-induced fluorescence. A separation medium composed of 1% HPMC in TBE buffer and 10 µmol/L intercalating dye (TOPRO-3) was demonstrated to resolve the amplicon of HCV with 135 by in length in less than 1.5 minutes with the confidence interval of the migration time less than 1.3%. Analysis of more than 10 patient samples by both the existing method using agarose gel electrophoresis and the current method using microchip electrophoresis showed a 100% correlation. The polymer microchip, with advantages including fast processing time, simple operation, and disposable use, holds great potential for clinical analysis.

Chip-based microfluidic devices coupled with electrospray ionization-mass spectrometry

Here we present an easy method to fabricate the poly (dimethylsiloxane) (PDMS)-based microfluidic chips with integrated functions for protein identification by tandem mass spectrometry using replica-molding techniques. This microchip has typical electrophoretic microchannels, a pulled nano-electrospray ionization (ESI) capillary tip at the outlet and a cartridge packed with the trypsin-immobilized beads attached to sample reservoir of the device. Moreover, a novel frit made-up by photopolymer technique was fabricated near the front of the separation channel, where the C18 beads were packed for de-salting. Using β-casein as a test protein, the MS spectra acquired from different migration times indicate that the digested fragments were separated. Moreover, some signature peptides of beta-casein were clearly identified by matching their detected molecular weights as well as sequences with those obtained from the database.