Evaluation of coloured materials in microfluidic flow-cells for chemiluminescence detection.

Recent advances in the construction of chemiluminescence flow-cells has included high precision milling of channels into a range of different polymer materials, in efforts to maximise the transfer of light from the chemical reaction to the photodetector. However, little is known of the extent that the colour of polymer materials will influence this transfer. This may become increasingly important as chemiluminescence detection zones are integrated with other operations within microfluidic devices or micro total analysis systems (µTAS). Herein, we compare microfluidic flow-cells fabricated from five polymer sheets (clear, white, black, red, blue), using two flow-cell designs (spiral and serpentine), two modes of photodetection, and four chemiluminescence reactions that provide a range of different emission colours. The direct transfer of light from the reaction within the white flow-cell channel to the photodetector made only minor contributions (10%-20%) to the measured intensity, with the majority of the measured light first interacting with the polymer material into which the channels were machined. The extent that the emitted light was absorbed or reflected by the coloured polymer materials was dependent on not only the properties of the polymer, but also the spectral distribution of the chemiluminescence. The changes in chemiluminescence intensities from absorption of light by the flow-cell materials can be accompanied by distortion of the spectral distribution.

Analytically useful blue chemiluminescence from a water-soluble iridium(III) complex containing a tetraethylene glycol functionalised triazolylpyridine ligand.

We examine [Ir(df-ppy)2(pt-TEG)](+) as the first highly water soluble, blue-luminescent iridium(III) complex for chemiluminescence detection. Marked differences in selectivity were observed between the new complex and the conventional [Ru(bpy)3](2+) reagent, which will enable this mode of detection to be extended to new areas of application.

Sex identification of ancient DNA samples using a microfluidic device.

Ancient DNA is the name given to the degraded, fragmented, and chemically damaged biomolecules that can be recovered from archaeological remains of plants, animals, and humans. Where ancient human DNA has survived at archaeological sites, it can give valuable information and is especially useful for its potential to identify kinship, population affinities, pathogens, and biological sex. Here, we describe the operation of a microfluidic device for the sex identification of ancient DNA samples using an efficient sample handling process. DNA is extracted from powdered bone samples and abasic sites labeled with biotin. Streptavidin-coated superparamagnetic particles are used to isolate the labeled DNA prior to amplification of the Amelogenin sex marker.

Advances in processes for PET radiotracer synthesis: separation of [¹8F]fluoride from enriched [¹8O]water.

Positron emission tomography (PET) is a powerful scientific and clinical tool for the study and visualization of human physiology that can provide important information about metabolism and diseases such as cancer. At present, [(18)F]fluorodeoxyglucose ([(18)F]FDG) is the most frequently used radiotracer for the routine clinical evaluation of malignant tumors in a range of body tissues. FDG synthesis is continuously being developed to improve and simplify the synthetic procedure including the isolation of [(18)F]fluoride from [(18)O]water. There are many methods reported in literature for the isolation of [(18)F]fluoride, including evaporation, coat-capture-elution, the use of cation-exchange resin and electrode trapping. This review article gives an overview of some of the most common methods for the separation of [(18)F]fluoride ions from [(18)O]water, highlighting the potential strength of the methods and also problems and weaknesses for synthesis of (18)F PET tracers.

Maternal hair selenium levels as a possible long-term nutritional indicator of recurrent pregnancy loss.

BACKGROUND: Approximately 1% of all couples trying to conceive will suffer from recurrent pregnancy loss (RPL). Nutritional deficiencies have been postulated as a possible cause of RPL and in particular, selenium deficiency has been associated with reproductive failure in animal studies and more recently, in some human studies. This study was undertaken to assess the maternal hair selenium levels in women with RPL without an identified cause and to compare these results with those of women with successful reproductive histories. METHODS: Twenty four patients with RPL and twenty four control subjects with at least one successful pregnancy and no pregnancy failures, who were matched for age and ethnicity, were recruited. A questionnaire was completed, which included demographic and social information and a dietary history. Hair samples were collected and analyzed for selenium content by inductively coupled plasma mass spectrometry. RESULTS: The control subjects had a higher mean income and had completed more years of education compared with the RPL patients. There was no significant difference in the intake of selenium rich foods between the 2 groups. The patients, however, consumed significantly more fruit, cheese, potatoes and chocolate than the controls. The median (range) selenium content was 0.80 ppm (0.19-4.15) and 0.68 ppm (0.43-3.76) in patients and controls respectively (Mann Whitney U test 209.5 p = 0.74). CONCLUSIONS: While there were significant differences in the 2 groups with regard to resources, education and diet our results show that hair selenium concentrations and dietary selenium intake, were similar in the two groups. Both groups had low levels of this important element.

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.

Antifungal synergy of theaflavin and epicatechin combinations against Candida albicans.

New antifungal agents are required to compensate for the increase in resistance to standard antifungal agents of Candida albicans, which is an important opportunistic fungal pathogen that causes minor infections in many individuals but very serious infections in those who are immune-compromised. In this study, combinations of theaflavin and epicatechin are investigated as potential antifungal agents and also to establish whether antifungal synergy exists between these two readily accessible and cost-effective polyphenols isolated from black and green tea. The results of disc diffusion assays showed stronger antibacterial activity of theaflavin:epicatechin combinations against C. albicans NCTC 3255 and NCTC 3179, than that of theaflavin alone. Minimum inhibitory concentrations (MICs) of 1,024 µg/ml with theaflavin and 128-256 µg/ml with theaflavin:epicatechin combinations were found. The fractional inhibitory concentration indexes were calculated, and the synergy between theaflavin and epicatechin against both isolates of C. albicans was confirmed. Theaflavin:epicatechin combinations show real potential for future use as a treatment for infections caused by C. albicans.

The application of microfluidic devices for viral diagnosis in developing countries.

Whilst diseases such as diabetes and cardiovascular disorders are increasing in the developed world, the main threat to global health remains viral-based infectious disease. Such diseases are notably prevalent in developing countries, where they represent a major cause of mortality; however, their detection and prevention is typically hampered by poor infrastructure and a lack of resources to support the sophisticated diagnostic tools commonly found in modern laboratories. Microfluidic-based diagnostics has the potential to close the gap between developed and developing world medical needs due to the robustness and reduced operating costs such technology offers. The most recent developments in microfluidic diagnostics for viral infections have explored the separation and enumeration of immune cells, the capture and identification of viral particles, and antiviral drug evaluation within microchannels and chambers. Advances in solid-phase separation, isothermal amplification, real-time detection of nucleotide products, and improved efficiency of detection systems in microfluidic platforms have also opened up opportunities for diagnostic innovation. This chapter reviews the potential capability microfluidic technology can offer in addressing the practical challenges of providing diagnostic technology for developing countries, illustrated by research on key viral diseases.

Improved DNA extraction efficiency from low level cell numbers using a silica monolith based micro fluidic device.

The evaluation of a micro fluidic system with an integrated silica monolith for performing DNA extraction from limited biological samples has been carried out. Low DNA target concentrations usually require the addition of carrier RNA to ensure desired extraction efficiencies. Here, we demonstrate a micro fluidic extraction system with increasingly efficient extraction performances for biological samples containing <15 ng of total DNA without the need of adding carrier nucleic acids. All extracted DNA showed successful amplification via the polymerase chain reaction demonstrating both the effectiveness of the proposed system at removing potential inhibitors and yielding good quality DNA. The work presented here beneficially identifies reduced sample volumes/concentrations as suitable for processing with respect to downstream analysis by enabling pre-concentration of the biological sample, particularly important when dealing with clinical or forensic specimens.

Evaluation of heart tissue viability under redox-magnetohydrodynamics conditions: toward fine-tuning flow in biological microfluidics applications.

A microfluidic system containing a chamber for heart tissue biopsies, perfused with Krebs-Henseleit buffer containing glucose and antibiotic (KHGB) using peristaltic pumps and continuously stimulated, was used to evaluate tissue viability under redox-magnetohydrodynamics (redox-MHD) conditions. Redox-MHD possesses unique capabilities to control fluid flow using ionic current from oxidation and reduction processes at electrodes in a magnetic field, making it attractive to fine-tune fluid flow around tissues for "tissue-on-a-chip" applications. The manuscript describes a parallel setup to study two tissue samples simultaneously, and 6-min static incubation with Triton X100. Tissue viability was subsequently determined by assaying perfusate for lactate dehydrogenase (LDH) activity, where LDH serves as an injury marker. Incubation with KHGB containing 5 mM hexaammineruthenium(III) (ruhex) redox species with and without a pair of NdFeB magnets (∼ 0.39 T, placed parallel to the chamber) exhibited no additional tissue insult. MHD fluid flow, viewed by tracking microbeads with microscopy, occurred only when the magnet was present and stimulating electrodes were activated. Pulsating MHD flow with a frequency similar to the stimulating waveform was superimposed over thermal convection (from a hotplate) for Triton-KHGB, but fluid speed was up to twice as fast for ruhex-Triton-KHGB. A large transient ionic current, achieved when switching on the stimulating electrodes, generates MHD perturbations visible over varying peristaltic flow. The well-controlled flow methodology of redox-MHD is applicable to any tissue type, being useful in various drug uptake and toxicity studies, and can be combined equally with on- or off-device analysis modalities.

A microfluidic system for testing the responses of head and neck squamous cell carcinoma tissue biopsies to treatment with chemotherapy drugs.

Tumors are heterogeneous masses of cells characterized pathologically by their size and spread. Their chaotic biology makes treatment of malignancies hard to generalize. We present a robust and reproducible glass microfluidic system, for the maintenance and "interrogation" of head and neck squamous cell carcinoma (HNSCC) tumor biopsies, which enables continuous media perfusion and waste removal, recreating in vivo laminar flow and diffusion-driven conditions. Primary HNSCC or metastatic lymph samples were subsequently treated with 5-fluorouracil and cisplatin, alone and in combination, and were monitored for viability and apoptotic biomarker release 'off-chip' over 7 days. The concentration of lactate dehydrogenase was initially high but rapidly dropped to minimally detectable levels in all tumor samples; conversely, effluent concentration of WST-1 (cell proliferation) increased over 7 days: both factors demonstrating cell viability. Addition of cell lysis reagent resulted in increased cell death and reduction in cell proliferation. An apoptotic biomarker, cytochrome c, was analyzed and all the treated samples showed higher levels than the control, with the combination therapy showing the greatest effect. Hematoxylin- and Eosin-stained sections from the biopsy, before and after maintenance, demonstrated the preservation of tissue architecture. This device offers a novel method of studying the tumor environment, and offers a pre-clinical model for creating personalized treatment regimens.

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.

Scaling up of continuous-flow, microwave-assisted, organic reactions by varying the size of Pd-functionalized catalytic monoliths.

A product-scalable, catalytically mediated flow system has been developed to perform Suzuki-Miyaura reactions under a microwave heating regime, in which the volumetric throughput of a Pd-supported silica monolith can be used to increase the quantity of the product without changing the optimal operating conditions. Two silica monoliths (both 3 cm long), with comparable pore diameters and surface areas, were fabricated with diameters of 3.2 and 6.4 mm to give volumetric capacities of 0.205 and 0.790 mL, respectively. The two monoliths were functionalized with a loading of 4.5 wt % Pd and then sealed in heat-shrinkable Teflon(®) tubing to form a monolithic flow reactor. The Pd-supported silica monolith flow reactor was then placed into the microwave cavity and connected to an HPLC pump and a backpressure regulator to minimize the formation of gas bubbles. The flow rate and microwave power were varied to optimize the reactant contact time and temperature, respectively. Under optimal reaction conditions the quantity of product could be increased from 31 mg per hour to 340 mg per hour simply by changing the volumetric capacity of the monolith.

Microscreening toxicity system based on living magnetic yeast and gradient chips.

There is an increasing demand for easy and cost-effective methods to screen the toxicological impact of the growing number of chemical mixtures being generated by industry. Such a screening method has been developed using viable, genetically modified green fluorescent protein (GFP) reporter yeast that was magnetically functionalised and held within a microfluidic device. The GFP reporter yeast was used to detect genotoxicity by monitoring the exposure of the cells to a well-known genotoxic chemical (methyl methane sulfonate, MMS). The cells were magnetised using biocompatible positively charged PAH-stabilised magnetic nanoparticles with diameters around 15 nm. Gradient mixing was utilised to simultaneously expose yeast to a range of concentrations of toxins, and the effective fluorescence emitted from the produced GFP was measured. The magnetically enhanced retention of the yeast cells, with their facile subsequent removal and reloading, allowed for very convenient and rapid toxicity screening of a wide range of chemicals. This is the first report showing magnetic yeast within microfluidic devices in a simple bioassay, with potential applications to other types of fluorescent reporter yeast in toxicological and biomedical research. The microfluidic chip offers a simple and low-cost screening test that can be automated to allow multiple uses (adapted to different cell types) of the device on a wide range of chemicals and concentrations.

Development of a real-world direct interface for integrated DNA extraction and amplification in a microfluidic device.

Integrated DNA extraction and amplification have been carried out in a microfluidic device using electro-osmotic pumping (EOP) for fluidic control. All the necessary reagents for performing both DNA extraction and polymerase chain reaction (PCR) amplification were pre-loaded into the microfluidic device following encapsulation in agarose gel. Buccal cells were collected using OmniSwabs [Whatman™, UK] and manually added to a chaotropic binding/lysis solution pre-loaded into the microfluidic device. The released DNA was then adsorbed onto a silica monolith contained within the DNA extraction chamber and the microfluidic device sealed using polymer electrodes. The washing and elution steps for DNA extraction were carried out using EOP, resulting in transfer of the eluted DNA into the PCR chamber. Thermal cycling, achieved using a Peltier element, resulted in amplification of the Amelogenin locus as confirmed using conventional capillary gel electrophoresis. It was demonstrated that the PCR reagents could be stored in the microfluidic device for at least 8 weeks at 4 °C with no significant loss of activity. Such methodology lends itself to the production of 'ready-to-use' microfluidic devices containing all the necessary reagents for sample processing, with many obvious applications in forensics and clinical medicine.

Microfluidic perfusion system for maintaining viable heart tissue with real-time electrochemical monitoring of reactive oxygen species.

A microfluidic device has been developed to maintain viable heart tissue samples in a biomimetic microenvironment. This device allows rat or human heart tissue to be studied under pseudo in vivo conditions. Effluent levels of lactate dehydrogenase and hydrogen peroxide were used as markers of damaged tissue in combination with in situ electrochemical measurement of the release of reactive oxygen species (ROS). The parameters for perfusion were optimized to maintain biopsies of rat right ventricular or human right atrial tissue viable for up to 5 and 3.5 hours, respectively. Electrochemical assessment of the oxidation current of total ROS, employing cyclic voltammetry, gave results in real-time that were in good agreement to biochemical assessment using conventional, off-chip, commercial assays. This proof-of-principle, integrated microfluidic device, may be exploited in providing a platform technology for future cardiac research, offering an alternative approach for investigating heart pathophysiology and facilitating the development of new therapeutic strategies.

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.

Microfluidic-based measurements of cytochrome P450 enzyme activity of primary mammalian hepatocytes.

A microfluidic-based system was developed for the in situ monitoring of the 7-ethoxyresorufin O-dealkylation (EROD) activity of primary rat hepatocytes by measuring the fluorescent intensity of both cells and their surrounding media. The microfluidic chip was designed to allow the cell suspension and test reagent to be introduced in a layer-by-layer flow format, thereby resulting in a short mixing time by diffusion. A good linear relationship was obtained between the resorufin concentration up to 30 microM and fluorescent intensity over the chip's circular chamber area. The EROD activity was determined with 3-methylcholanthrene (3-MC)-induced hepatocytes. The inhibition effect of alpha-naphthoflavone was also examined on EROD activity resulting in an IC(50) value of 12.98 microM.

Detection of nitric oxide in tissue samples by ESI-MS.

A novel method to determine nitric oxide (NO) in biological tissue samples with minimal interference from the cellular detritus is described. Methylpiperazinobenzenediamine, consisting of an o-phenylenediamine and a methyl piperazine group, was chosen as a probe for the detection of NO by mass spectrometry (MS) in biological tissue samples. The o-phenylenediamine group reacts with NO to form a characteristic benzotriazole. The product was identified using electrospray ionization mass spectrometry (ESI-MS) and the method validated within the range of 95-1900 nM. NO levels associated with tissue biopsies (approximately 10 mg) from rat vasculature and intestine tissue biopsies have been successfully determined. The different rates of NO generated from tissue samples under hypoxic and normoxic conditions have been studied by this simple and sensitive method.

Measurement of acetylcholinesterase inhibition using bienzymes immobilized monolith micro-reactor with integrated electrochemical detection.

This paper reports a simple micro-FIA based method for the rapid evaluation of acetylcholinesterase inhibition based on bienzymes immobilized monolith micro-reactor, with integrated electrochemical detection. The monolith was prepared inside a micro-fluidic device from two precursors TMOS and MTMOS using a sol-gel method, followed by PEI polymer functionalization and subsequent enzyme immobilization via electrostatic attraction between electronegative enzymes and electropositive PEI polymers. A bienzyme system containing co-immobilized acetylcholinesterase and choline oxidase was used for the evaluation of enzyme inhibition induced by malaoxon, eserine and methomyl analytes. The proposed method, which gave a LOD of 0.5, 0.2 and 1.0 microM for malaoxon, eserine and methomyl repeatedly, was found to offer several advantages over existing systems including efficient enzyme immobilization, minimal reagent consumption and rapid analysis capability.