Integrated RNA extraction and RT-PCR for semi-quantitative gene expression studies on a microfluidic device

This paper describes the development of a microfluidic methodology, using RNA extraction and reverse transcription PCR, for investigating expression levels of cytochrome P450 genes. Cytochrome P450 enzymes are involved in the metabolism of xenobiotics, including many commonly prescribed drugs, therefore information on their expression is useful in both pharmaceutical and clinical settings. RNA extraction, from rat liver tissue or primary rat hepatocytes, was performed using a silica-based solid-phase extraction technique. Following elution of the purified RNA, amplification of target sequences for the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the cytochrome P450 gene CYP1A2, was carried out using a one-step reverse transcription PCR. Once the microfluidic methodology had been optimized, analysis of control and 3-methylcholanthrene-induced primary rat hepatocytes were used to evaluate the system. As expected, GAPDH was consistently expressed, whereas CYP1A2 levels were found to be raised in the drug-treated samples. The proposed system offers an initial platform for development of both rapid throughput analyzers for pharmaceutical drug screening and point-of-care diagnostic tests to aid provision of drug regimens, which can be tailor-made to the individual patient.

Integrated DNA extraction and amplification using electrokinetic pumping in a microfluidic device

An integrated system employing anion exchange for the extraction of DNA from biological samples prior to polymerase chain reaction DNA amplification has been developed, based on microfluidic methodology utilising electrokinetic pumping. In this system, the biological samples were added directly to chitosan-coated silica beads to facilitate DNA immobilisation. The purified, pre-concentrated DNA was then eluted using a combination of electro-osmotic flow enhanced with electrophoretic mobility, which enable DNA to be transported by both mechanisms into the DNA amplification chamber. Through optimisation of the DNA elution conditions, average DNA extraction efficiencies of 69.1% were achievable. Subsequent DNA amplification performed on the microfluidic system demonstrated not only the ability to use electrokinetic movement to integrate the two processes on a single device, but also that the quality and quantity of DNA eluted was suitable for downstream analysis. This work offers an attractive real-world to chip interface and a route to simpler Lab-on-a-Chip technology which eliminates the need for moving parts.

Direct processing of clinically relevant large volume samples for the detection of sexually transmitted infectious agents from urine on a microfluidic device

Urine is a preferred specimen for nucleic acid-based detection of sexually transmitted infections (STIs) but represents a challenge for microfluidic devices due to low analyte concentrations. We present an extraction methodology enabling rapid on-chip nucleic acid purification directly from clinically relevant sample volumes up to 1 ml and subsequent PCR amplification detection.

Design and properties of a new hybrid stent-graft device

This PhD work explored a novel bio-inspired approach for designing artificial blood vessel implants known as stent-grafts. The design was inspired from body design of a caterpillar. This design concept induced natural flexibility and expandability property in the new stent-graft, which is considered critical in deciding long-term health of treated patients.