Charlotte E. Dyer

Rapid PCR amplification using a microfluidic device with integrated microwave heating and air impingement cooling

A microwave heating system is described for performing polymerase chain reaction (PCR) in a microfluidic device. The heating system, in combination with air impingement cooling, provided rapid thermal cycling with heating and cooling rates of up to 65 degrees C s(-1) and minimal over- or under-shoot (+/-0.1 degrees C) when reaching target temperatures. In addition, once the required temperature was reached it could be maintained with an accuracy of +/-0.1 degrees C. To demonstrate the functionality of the system, PCR was successfully performed for the amplification of the Amelogenin locus using heating rates and quantities an order of magnitude faster and smaller than current commercial instruments.

Development of a microfluidic device for the maintenance and interrogation of viable tissue biopsies

A microfluidic based experimental methodology has been developed that offers a biomimetic microenvironment in which pseudo in vivo tissue studies can be carried out under in vitro conditions. Using this innovative technique, which utilizes the inherent advantages of microfluidic technology, liver tissue has been kept in a viable and functional state for over 70 h during which time on-chip cell lysis has been repeatedly performed. Tissue samples were also disaggregated in situ on-chip into individual primary cells, using a collagenase digestion procedure, enabling further cell analysis to be carried out off-line. It is anticipated that this methodology will have a wide impact on biological and clinical research in fields such as cancer prognosis and treatment, drug development and toxicity, as well as enabling better fundamental research into tissue/cell processes.

The use of carrier RNA to enhance DNA extraction from microfluidic-based silica monoliths

DNA extraction was carried out on silica-based monoliths within a microfluidic device. Solid-phase DNA extraction methodology was applied in which the DNA binds to silica in the presence of a chaotropic salt, such as guanidine hydrochloride, and is eluted in a low ionic strength solution, such as water. The addition of poly-A carrier RNA to the chaotropic salt solution resulted in a marked increase in the effective amount of DNA that could be recovered (25ng) compared to the absence of RNA (5ng) using the silica-based monolith. These findings confirm that techniques utilising nucleic acid carrier molecules can enhance DNA extraction methodologies in microfluidic applications.

A microfluidic device for tissue biopsy culture and interrogation

This communication reports the development of a microfluidic device capable of maintaining the long-term culture of viable tissue biopsies. Tissue-based models will enable evaluation of cell–cell and cell–matrix interactions within multi-cellular systems. The device demonstrated is a prototype, fabricated with the capacity to receive biopsy samples up to 2 mm3, from various tissue sources. Presently, this system has been tested with human colorectal tissue biopsies, for periods in excess of 3 days. The response of normal colorectal tissue and neoplastic biopsies to hypoxia was assayed by the release of vascular endothelial growth factor (VEGF) into the media, which was measured off-chip. As anticipated, the hypoxia induced a greater VEGF response in the tumour biopsies than the non-malignant tissue.

Development of a bi-functional silica monolith for electro-osmotic pumping and DNA clean-up/extraction using gel-supported reagents in a microfluidic device

A silica monolith used to support both electro-osmotic pumping (EOP) and the extraction/elution of DNA coupled with gel-supported reagents is described. The benefits of the combined EOP extraction/elution system were illustrated by combining DNA extraction and gene amplification using the polymerase chain reaction (PCR) process. All the reagents necessary for both processes were supported within pre-loaded gels that allow the reagents to be stored at 4 degrees C for up to four weeks in the microfluidic device. When carrying out an analysis the crude sample only needed to be hydrodynamically introduced into the device which was connected to an external computer controlled power supply via platinum wire electrodes. DNA was extracted with 65% efficiency after loading lysed cells onto a silica monolith. Ethanol contained within an agarose gel matrix was then used to wash unwanted debris away from the sample by EOP (100 V cm(-1) for 5 min). The retained DNA was subsequently eluted from the monolith by water contained in a second agarose gel, again by EOP using an electric field of 100 V cm(-1) for 5 min, and transferred into the PCR reagent containing gel. The eluted DNA in solution was successfully amplified by PCR, confirming that the concept of a complete self-contained microfluidic device could be realised for DNA sample clean up and amplification, using a simple pumping and on-chip reagent storage methodology.

Simple practical approach for sample loading prior to DNA extraction using a silica monolith in a microfluidic device

A novel DNA loading methodology is presented for performing DNA extraction on a microfluidic system. DNA in a chaotropic salt solution was manually loaded onto a silica monolith orthogonal to the subsequent flow of wash and elution solutions. DNA was successfully extracted from buccal swabs using electro-osmotic pumping (EOP) coupled with in situ reagents contained within a 1.5% agarose gel matrix. The extracted DNA was of sufficient quantity and purity for polymerase chain reaction (PCR) amplification.

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.

Tumour-expressed tissue factor inhibits cellular cytotoxicity

Aims: The association between tissue factor (TF) expression and increased rate of tumour metastasis is well established. In this study, we have examined the hypothesis that the expression of TF by disseminated tumour cells confers protection against immune recognition and cytotoxicity. Materials and methods: A hybrid EGFP-TF protein was expressed in HT29 colon carcinoma and K562 lymphoblast cell lines. To assess the cytotoxic activity against tumour cells over-expressing TF, a novel method was used, based on the direct measurement of fluorescently labelled HT29 or K562 target cells. Results: Upon challenge with peripheral blood mononuclear cells (PBMC), tumour cells expressing TF partially evaded cellular cytotoxicity (Δ=15–40% reduction in cytotoxicity). Moreover, the influence of TF was not primarily dependent on its procoagulant function, although the inclusion of 20% (v/v) plasma did lower the rate of cytotoxicity against untransfected cells. However, expression of a truncated form of TF, devoid of the cytoplasmic domain, did not mediate any degree of inhibition of cytotoxicity, suggesting that the protective function of TF is principally due to this domain. Conclusions: We conclude that TF can promote immune evasion in tumour cells expressing this protein leading to increased survival and therefore metastatic rate in such cells.

On-chip integrated labelling, transport and detection of tumour cells†

Microflow cytometry represents a promising tool for the investigation of diagnostic and prognostic cellular cancer markers, particularly if integrated within a device that allows primary cells to be freshly isolated from the solid tumour biopsies that more accurately reflect patient‐specific in vivo tissue microenvironments at the time of staining. However, current tissue processing techniques involve several sequential stages with concomitant cell losses, and as such are inappropriate for use with small biopsies. Accordingly, we present a simple method for combined antibody‐labelling and dissociation of heterogeneous cells from a tumour mass, which reduces the number of processing steps. Perfusion of ex vivo tissue at 4°C with antibodies and enzymes slows cellular activity while allowing sufficient time for the diffusion of minimally active enzymes. In situ antibody‐labelled cells are then dissociated at 37°C from the tumour mass, whereupon hydrogel‐filled channels allow the release of relatively low cell numbers (<1000) into a biomimetic microenvironment. This novel approach to sample processing is then further integrated with hydrogel‐based electrokinetic transport of the freshly liberated fluorescent cells for downstream detection. It is anticipated that this integrated microfluidic methodology will have wide‐ranging biomedical and clinical applications.

Apolipoprotein E ε4 Allele Modulates the Immediate Impact of Acute Exercise on Prefrontal Function

The difference between Apolipoprotein E ε4 carriers and non-carriers in response to single exercise sessions was tested. Stroop and Posner tasks were administered to young untrained women immediately after walking sessions or moderately heavy exercise. Exercise had a significantly more profound impact on the Stroop effect than on the Posner effect, suggesting selective involvement of prefrontal function. A significant genotype-by-exercise interaction indicated differences in response to exercise between ε4 carriers and non-carriers. Carriers showed facilitation triggered by exercise. The transient executive down-regulation was construed as due to exercise-dependent hypofrontality. The facilitation observed in carriers was interpreted as better management of prefrontal metabolic resources, and explained within the antagonistic pleiotropy hypothesis framework. The findings have implications for the interpretation of differences between ε4 carriers and non-carriers in the benefits triggered by long-term exercise that might depend, at least partially, on mechanisms of metabolic response to physical activity.