DNA oligonucleotide synthesis in a microdevice for multiple analytical applications

Abstract

DNA oligonucleotide, a short piece of DNA, is one of the most commonly used materials in biomolecular applications. Nowadays, most oligonucleotides are synthesized in commercialized oligonucleotide synthesizers. Due to the complexity of the synthesis process, these synthesizers are bulky in size. Recently, there is an emerging need of on-site applications of oligonucleotides, such as in civil defense to immediately detect biological attacks. However, due to the size limitation of the available oligonucleotide synthesizers, only pre-made oligonucleotides with certain sequence can be brought to the field. It is practically impossible to get oligonucleotide of any sequence on demand in the field. In this thesis, a solution is offered by proposing and building a portable oligonucleotide synthesizer based on microfluidic technology.

Firstly, a microfluidic chip with integrated microvalves is developed. The microvalves have zero dead-volume characteristic that is attributed to the design of zigzag shaped main channel and special position of each microvalve. This design removes the connecting channels between the microvalve and the main channel that are usually found in traditional designs. Therefore, reagents cannot be trapped within the microfluidic device and cross contamination can be effectively minimized. The zero dead-volume characteristic is proven by detection of trace amount of DNA template molecules trapped in the device. This contamination free characteristic is extremely important for applications, such as DNA oligonucleotide synthesis, in which cross contamination is critical issue and can lead to failure of the synthesis.

Secondly, a portable oligonucleotide synthesizer based on the developed zero dead-volume microchip is built. The portable synthesizer has the ability to synthesize oligonucleotide as either primer or probe. The sequence and biological functionality of the synthesized oligonucleotides are proven by polyacrylamide gel electrophoresis, PCR and DNA hybridization experiment. The portable synthesizer has the ability to synthesize oligonucleotide of any sequence on demand. To the best of our knowledge, this is the first reported portable oligonucleotide synthesizer.

To further improve the function of the portable system and realize generic DNA bioassay within it, a DNA hybridization and fluorescence detection unit is presented and integrated into the portable synthesizer. The fully integrated system, a portable generic DNA hybridization bioassay system, is successfully used to distinguish different bacteria strains based on in situ DNA oligonucleotide synthesis, hybridization and fluorescence detection. As far as we know, this is also the first portable generic DNA bioassay system reported. As the system has the potential to detect DNA target of any sequence in the field, we envisage that the system could help to enable fast responses to emerging bio-threats for homeland security and in pandemics.