Bead-based microfluidic platform for biochemical applications
e are developing a microfluidic flow-through system for miniaturized DNA pyrosequencing. Pyrosequencing is a technique based on the detection of released pyrophosphate during DNA synthesis.
In recent years, microfabricated systems capable of performing biological analysis have been the subject of numerous studies. Using standard photolithographic techniques, network of micrometer-sized channels can be etched into a variety of substrates, most frequently glass or silicon.
Previously, we presented a microfluidic flow-through device for biochemical reactions on beads. A microfluidic approach for rapid bioluminescent real-time detection of single nucleotide polymorphism (SNP) has successfully been performed in the microfluidic flow-through device .
Today, we are developing a microfluidic flow-through system for miniaturized DNA pyrosequencing. Pyrosequencing is a technique based on the detection of released pyrophosphate during DNA synthesis. A schematic draw of the principle of pyrosequencing is shown in the figure.
The microfluidic platform allowed detection of single base incorporation, enabling base-by-base DNA sequencing. Result of single nucleotide incorporation in the microfluidic device is shown in Figure 2. The reaction chamber containing the DNA template volume is 12.5 - 50 nl and the reagent volume dispensed was 100-200 nl. This can be compared with 10–50 µl used in commercial Pyrosequencing™ instruments manufactured by Pyrosequencing AB.
The figure above shows the result of a single nucleotide incorporation in the microfluidic device.
The combined use of functionalized particles and MEMS structures is a powerful analytical tool. A novel technique for self-assembly and immobilization of nano- and microparticles has been developed based on surface chemistry. It is a fast, convenient and simple method, involving microcontact printing and self-assembly, that can be applied to unstructured and structured glass and silicon.
Currently we are working on miniaturizing a new SNP scoring method, called dynamic allele-specific hybridization (DASH), onto immobilized monolayers of patterned beads on chip. The DASH assay is in essence a dynamic heating of DNA target-probe samples and the monitoring of their denaturation with information coming from differences in melting points between matched and mismatched DNA. Microcontact printing using elastomere poly(dimethylsiloxane) (PDMS) stamp was employed to create patterned monolayers of beads containing target DNA-probe duplex on a silicondioxide surface. The patterned monolayers of beads are heated through and beyond the melting temperature of the duplexes. In order to have complete localized control over the temperature and obtaining higher temperature resolution, internal heater and temperature sensors are integrated on the chip. The bead-based DASH procedure enables dramatic reduction of the working volumes and offers higher temperature resolution which is a step towards developing cost-effective high-throughput DASH method on arrays of single beads.
- SSF through the Nanochemistry program Vinnova
Publications related to this project