Chia-Sheng Liao

Integrated microfluidic systems for cell lysis, mixing/pumping and DNA amplification

The present paper reports a fully automated microfluidic system for the DNA amplification process by integrating an electroosmotic pump, an active micromixer and an on-chip temperature control system. In this DNA amplification process, the cell lysis is initially performed in a micro cell lysis reactor. Extracted DNA samples, primers and reagents are then driven electroosmotically into a mixing region where they are mixed by the active micromixer. The homogeneous mixture is then thermally cycled in a micro-PCR (polymerase chain reaction) chamber to perform DNA amplification. Experimental results show that the proposed device can successfully automate the sample pretreatment operation for DNA amplification, thereby delivering significant time and effort savings. The new microfluidic system, which facilitates cell lysis, sample driving/mixing and DNA amplification, could provide a significant contribution to ongoing efforts to miniaturize bio-analysis systems by utilizing a simple fabrication process and cheap materials.

Miniature RT–PCR system for diagnosis of RNA-based viruses

This paper presents an innovative portable chip-based RT–PCR system for amplification of specific nucleic acid and detection of RNA-based viruses. The miniature RT–PCR chip is fabricated using MEMS (Micro-electro-mechanical-system) techniques, and comprises a micro temperature control module and a PDMS (polydimethylsiloxane)-based microfluidic control module. The heating and sensing elements of temperature control module are both made of platinum and are located within the reaction chambers in order to generate a rapid and uniform thermal cycling. The microfluidic control module is capable of automating testing process with minimum human intervention. In this paper, the proposed miniature RT–PCR system is used to amplify and detect two RNA-based viruses, namely dengue virus type-2 and enterovirus 71 (EV 71). The experimental data confirm the ability of the system to perform a two-step RT–PCR process. The developed miniature system provides a crucial tool for the diagnosis of RNA-based viruses.

Circulating polymerase chain reaction chips utilizing multiple-membrane activation*

This paper reports a new micromachined, circulating, polymerase chain reaction (PCR) chip for nucleic acid amplification. The PCR chip is comprised of a microthermal control module and a polydimethylsiloxane (PDMS)-based microfluidic control module. The microthermal control modules are formed with three individual heating and temperature-sensing sections, each modulating a specific set temperature for denaturation, annealing and extension processes, respectively. Micro-pneumatic valves and multiple-membrane activations are used to form the microfluidic control module to transport sample fluids through three reaction regions. Compared with other PCR chips, the new chip is more compact in size, requires less time for heating and cooling processes, and has the capability to randomly adjust time ratios and cycle numbers depending on the PCR process. Experimental results showed that detection genes for two pathogens, Streptococcus pyogenes (S. pyogenes, 777 bps) and Streptococcus pneumoniae (S. pneumoniae, 273 bps), can be successfully amplified using the new circulating PCR chip. The minimum number of thermal cycles to amplify the DNA-based S. pyogenes for slab gel electrophoresis is 20 cycles with an initial concentration of 42.5 pg µl−1. Experimental data also revealed that a high reproducibility up to 98% could be achieved if the initial template concentration of the S. pyogenes was higher than 4 pg µl−1.

Microfluidic chips for DNA amplification, electrophoresis separation and on-line optical detection

This paper reports at innovative microfluidic chip capable of performing DNA amplification (polymerase chain reaction, PCR), electrokinetic sample injection and separation, and on-line optical detection of DNA. All microfluidic modules are integrated on cheap and biocompatible soda-lime glass substrates using a simple and reliable fabrication process. With this approach, DNA samples could be first duplicated using the micro PCR module, then injected and separated in the micro electrophoretic channels driven by electrokinetic forces, and finally detected optically by buried optical waveguides downstream the separation channel. In order to have a better separation efficiency for DNA samples, a novel surface treatment method by coating a thin layer of spin-on-glass (SOG) is developed. Experimental data show that DNA samples can be successfully duplicated using the micro PCR device with less sample and reagent volumes in a shorter period thanks to lower thermal inertia of the micro devices. DNA samples can be injected, separated and detected successfully in the subsequent microfluidic channels. The integrated microfluidic device could be crucial for genetic analysis.

Micromachined Flow-Through Polymerase Chain Reaction Chip Utilizing Multiple Membrane-Activated Micropumps

This paper reports a new micromachined flow-through polymerase chain reaction (PCR) chip for applications of rapid pathogen diagnosis. The PCR chip comprised a micro thermal control module and a microfluidic control module fabricated using MEMS (micro-electro-mechanical-systems) technology. The micro thermal control module was formed with three individual heating and temperature-sensing sections, each modulating a specific temperature for denaturation, annealing and extension process, respectively. The membrane-activated micropumps were used to transport sample fluids through three reaction regions to adjust the time ratio and cycle numbers for PCR. The experimental results showed that S. pneumoniae detection gene (273 bps) could be amplified successfully using the new flow-through PCR chip. The new PCR chip could be promising for rapid clinical diagnosis of DNA-based infectious disease.

Integrated Microfluidic Systems for DNA Analysis

This study reports the integration of an electrokinetically-driven micro-mixer with a on-chip temperature control system, and applies the integrated microfluidic chip to the DNA amplification process. Using the integrated chip, the cultured cells are initially broken down in a microanalysis reactor. Extracted DNA, primers and reagents are then driven electroosmotically into a mixing region where they are mixed by an electrokinetically-driven micro-mixer. The mixture is then cycled in a micro-PCR (polymerase chain reaction) chamber to perform DNA amplification. Experimental results show that the proposed device can automate the sample pretreatment operation for DNA amplification, thereby achieving significant time and effort savings. This novel integrated microfluidic device, which facilitates cell analysis, sample driving/mixing, and DNA amplification, could make a promising contribution to the continuing efforts aimed at miniaturizing bio-analysis systems

A New Self-Compensated Thermocycler for Polymerase Chain Reaction

This paper presents a new self-compensated thermocycler for polymerase chain reaction (PCR), especially used in DNA amplification process requiring precise thermal control. A new design of micro- heaters to improve the thermal uniformity of the PCR chamber using array-type heaters and a self- compensated mechanism is reported. The feature of this design is that structure change or active thermal compensation is not required. Experimental data show that the spatial variation of temperature distribution is less than ldegC in the PCR chamber at annealing temperature. Besides, the area with thermal variation less than 0.5degC is close to 90 % of the working area.

A fully-integrated microfluidic chip for RNA-virus detection

The study presents a new fully-integrated microfluidic chip capable of performing reverse transcription polymerase chain reaction (RT-PCR) (Obeid et al., 2003), transportation of DNA/RNA samples, capillary electrophoresis (CE) separation and on-line detection. In the proposed device, dengue-2 RNA-virus was synthesized to complementary DNA (cDNA) and replicated using a RT-PCR module and then transported by a pneumatic micropump to CE sample reservoir. The cDNA samples were separated electrophoretically and detected by buried optical fibers. We have successfully demonstrated the detection of a RNA-virus using less time and less consumption of samples and reagents. The developed microfluidic chip could provide a powerful tool for fast disease diagnosis.