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

Abstract

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.

Figure 1

(a) The operating principle of cell lysis by applying an electric field. When a cell is exposed to a high electric field, the trans-membrane potential (opposite charges on the inner and outer membranes) can be induced. When the induced trans-membrane potential is higher than a certain value (about 1 V), it may cause the cells to be disrupted. After the cell lysis process, the magnetic beads either with (b) positive charges or (c) nucleotides probes are used in the microfluidic system to perform the nucleic acid extraction for molecular diagnosis

Figure 2

Concepts and equivalent circuit of array-type heaters. (a) The grid pattern of array-type thermocycler is the heating power of each array-type heating grid. (b) Equivalent circuit for the grid array pattern. I is current, V is supplying voltage, and R is resistance of the original heating grid

Figure 3

Schematic illustration of the flow-through PCR chip with multiple membrane activation (Wang et al., Copyright 2007 IOP Science) and suction-type membrane activation (Chien et al., Copyright 2009 Springer)

Figure 4

A photograph of the microfluidic chips capable of DNA/RNA amplification, electrophoretic injection and separation and on-line detection of DNA samples. First, the integrated microfluidic chip can perform two-step RT-PCR using pneumatic micropumps to transport RT-PCR/PCR reagents (reservoirs 2 and 3) to the PCR chamber (reservoir 1). The RT-PCR chamber performs the reverse transcription reaction on mRNA. Precise amounts of RNA reagents/templates can be first transported to the neighboring “PCR chamber” from the “RT-PCR reagent chamber” for reverse transcription of RNA templates using pneumatic micropumps. After synthesis, complimentary DNA (cDNA) samples can be further amplified after pumping PCR reagents from the neighboring “PCR reagent chamber”. The two-step RT-PCR amplification process provides a more reliable method for genetic identification. If only DNA samples are to be amplified, then the reverse transcription process can be omitted. Secondly, these pneumatic micropumps could also act as microvalves such that PCR reagents and CE buffers could be properly separated (Huang et al., Copyright 2006 Wiley-Blackwell)