Jung-Hao Wang

Nucleic acid amplification using microfluidic systems

In the post-human-genome-project era, the development of molecular diagnostic techniques has advanced the frontiers of biomedical research. Nucleic-acid-based technology (NAT) plays an especially important role in molecular diagnosis. However, most research and clinical protocols still rely on the manual analysis of individual samples by skilled technicians which is a time-consuming and labor-intensive process. Recently, with advances in microfluidic designs, integrated micro total-analysis-systems have emerged to overcome the limitations of traditional detection assays. These microfluidic systems have the capability to rapidly perform experiments in parallel and with a high-throughput which allows a NAT analysis to be completed in a few hours or even a few minutes. These features have a significant beneficial influence on many aspects of traditional biological or biochemical research and this new technology is promising for improving molecular diagnosis. Thus, in the foreseeable future, microfluidic systems developed for molecular diagnosis using NAT will become an important tool in clinical diagnosis. One of the critical issues for NAT is nucleic acid amplification. In this review article, recent advances in nucleic acid amplification techniques using microfluidic systems will be reviewed. Different approaches for fast amplification of nucleic acids for molecular diagnosis will be highlighted.

A microfabricated CE chip for DNA pre-concentration and separation utilizing a normally closed valve.

A simple, sequential DNA pre‐concentration and separation method by using a micro‐CE chip integrated with a normally closed valve, which is activated by pneumatic suction, has been developed. The CE chip is fabricated using PDMS. A surface treatment technique for coating a polymer bilayer with an anionic charge is applied to modify the surface of the microchannel. A normally closed valve with anionic surface charges forms a nanoscale channel that only allows the passage of electric current but traps the DNA samples so that they are pre‐concentrated. After the pre‐concentration step, a pneumatic suction force is applied to the normally closed valve. This presses down the valve membrane, which reconnects the channels. The DNA samples are then moved into a separation channel for further separation and analysis. Successful DNA pre‐concentration and separation has been achieved. Fluorescent intensity at the pre‐concentration area is increased by approximately 3570 times within 1.9 min of operation. The signals from the separation of ϕX174 DNA/HaeIII markers are enhanced approximately 41 times after 100 s of pre‐concentration time, as compared with the results using a traditional cross‐shaped micro‐CE chip. These results clearly demonstrate that successful DNA pre‐concentration for signal enhancement and separation analysis can be performed by using this new micro‐CE chip.

Sample pretreatment and nucleic acid-based detection for fast diagnosis utilizing microfluidic systems.

Recently, micro-electro-mechanical-systems (MEMS) technology and micromachining techniques have enabled miniaturization of biomedical devices and systems. Not only do these techniques facilitate the development of miniaturized instrumentation for biomedical analysis, but they also open a new era for integration of microdevices for performing accurate and sensitive diagnostic assays. A so-called “micro-total-analysis-system”, which integrates sample pretreatment, transport, reaction, and detection on a small chip in an automatic format, can be realized by combining functional microfluidic components manufactured by specific MEMS technologies. Among the promising applications using microfluidic technologies, nucleic acid-based detection has shown considerable potential recently. For instance, micro-polymerase chain reaction chips for rapid DNA amplification have attracted considerable interest. In addition, microfluidic devices for rapid sample pretreatment prior to nucleic acid-based detection have also achieved significant progress in the recent years. In this review paper, microfluidic systems for sample preparation, nucleic acid amplification and detection for fast diagnosis will be reviewed. These microfluidic devices and systems have several advantages over their large-scale counterparts, including lower sample/reagent consumption, lower power consumption, compact size, faster analysis, and lower per unit cost. The development of these microfluidic devices and systems may provide a revolutionary platform technology for fast sample pretreatment and accurate, sensitive diagnosis.

Hepatitis B surface antigen induces an early‐type hypersensitivity

In addition to the delayed‐type hypersensitivity (DTH), a unique type of hypersensitivity could be induced at a late stage of the immune responses after hepatitis B surface antigen (HBsAg) immunization. This antigen‐specific ear swelling that develops within 1 h after antigen challenge has been referred to as the early‐type hypersensitivity (ETH) in contrast to the 24‐h DTH. Although expression of ETH was earlier than DTH, the induction of the former needed 3 days longer than that of the latter. In ETH, the plasma protein leaked into the tissue and the vasopermeability increased within 15 min, causing the oedema of ETH. The observation that cyproheptadine, not dexametha‐sone, inhibited ETH suggests that it is mediated through the release of histamine and/or serotonin. Furthermore, ETH could be transferred by immune sera. Heat treatment (56°C for 4 h) did not destroy the transfer, suggesting that it was not mediated by IgE. The human anti‐HBs sera from either hepatitis B virus infection or HBsAg vaccinee also contained the activity to transfer the ETH in mice

Formation of Tunable, Emulsion Micro-Droplets Utilizing Flow-Focusing Channels and a Normally-Closed Micro-Valve

A mono-dispersed emulsion is of great significance in many chemical, biomedical and industrial applications. The current study reports a new microfluidic chip capable of forming tunable micro-droplets in liquids for emulsification applications. It can precisely generate size-tunable, uniform droplets using flow-focusing channels and a normally-closed valve, which is opened by a pneumatic suction force. Experimental data showed that micro-droplets with a diameter ranging from several to tens of micrometers could be precisely generated with a high uniformity. The droplet size is experimentally found to be dependent on the velocity of the dispersed-phase liquid, which is controlled by the deflection of the suction membrane. Emulsions with droplet sizes ranging from 5.5 to 55 μm are successfully observed. The variation in droplet sizes is from 3.8% to 2.5%. The micro-droplets have a uniform size and droplets smaller than those reported in previous studies are possible with this approach. This new microfluidic device can be promising for emulsification and other related applications.

Integrated microfluidic chip for measuring T helper cells

This paper reports a new microfluidic chip capable of measuring and counting CD3/CD4 T helper cells. A T-cell count was essential for diagnosis of immunodeficiency and lymphocytic diseases. To achieve the measurement and counting of T helper cells, a new microfluidic device comprising a micro incubation and a micro flow cytometer module was presented in this study for sample pretreatment, measuring, and counting. First, the CD3/CD4 T helper cells and anti-human CD3/CD4 antibodies conjugated fluorescent proteins were co-incubated in the micro incubation. When the T helper cells flew through the micro flow cytometer, the intensity of fluorescent signals was then detected. The T helper cells would be counted unless both signals of anti-human CD3 and CD4 antibodies were induced at the same time. Thus, the specificity of T helper cells was measured to be 0.96. The developed microfluidic chip may provide a promising tool for fast monitoring of immunodeficiency and lymphocytic diseases.

A micro-fabricated capillary electrophoresis chip for DNA pre-concentration and separation

A simple online DNA pre-concentration and separation method by using a micro capillary electrophoresis (CE) chip integrated with a normally-closed valve activated by pneumatic suction has been developed. The normally-closed valve with anionic surface charges forms a nanoscale channel only allows the passage of electric current but traps the DNA samples so that they can be pre-concentrated. Obvious fluorescent-labeled DNA concentration is observed at the pre-concentration area. The fX174 DNA/HaeIII markers separation signals are significantly enhanced comparing with the results of a traditional cross-shaped micro CE chip. Therefore, successful DNA pre-concentration for signal enhancement and separation analysis can be performed by using this new micro CE chip.