We demonstrate the application of a multilaminar flow platform, in which functionalized magnetic particles are deflected through alternating laminar flow streams of reagents and washing solutions via an external magnet, for the rapid detection of the inflammatory biomarker, C-reactive protein (CRP). The two-step sandwich immunoassay was accomplished in less than 60 s, a vast improvement on the 80-300 min time frame required for enzyme-linked immunosorbent assays (ELISA) and the 50 min necessary for off-chip magnetic particle-based assays. The combination of continuous flow and a stationary magnet enables a degree of autonomy in the system, while a detection limit of 0.87 ?g mL(-1) makes it suitable for the determination of CRP concentrations in clinical diagnostics. Its applicability was further proven by assaying real human serum samples and comparing those results to values obtained using standard ELISA tests.
The tumour microenvironment is a highly complex region where multiple interactions occur between host and cancer cells. The host response is defined by the presence and function of different tumour infiltrating lymphocytes and cytokines. Head and neck squamous cell carcinomas comprise a subgroup of human cancers with significant morbidity and mortality.
Objective The aim of this study was to investigate how head and neck squamous cell carcinoma (HNSCC) tissue biopsies maintained in a pseudo in vivo environment within a bespoke microfluidic device respond to radiation treatment. Study Design Feasibility study. Setting Tertiary referral center. Subjects and Methods Thirty-five patients with HNSCC were recruited, and liver tissue from 5 Wistar rats was obtained. A microfluidic device was used to maintain the tissue biopsy samples in a viable state. Rat liver was used to optimize the methodology. HNSCC was obtained from patients with T1-T3 laryngeal or oropharyngeal SCC; N1-N2 metastatic cervical lymph nodes were also obtained. Irradiation consisted of single doses of between 2 Gy and 40 Gy and a fractionated course of 5×2 Gy. Cell death was assessed in the tissue effluent using the soluble markers lactate dehydrogenase (LDH) and cytochrome c and in the tissue by immunohistochemical detection of cleaved cytokeratin18 (M30 antibody). Results A significant surge in LDH release was demonstrated in the rat liver after a single dose of 20 Gy; in HNSCC, it was seen after 40 Gy compared with the control. There was no significant difference in cytochrome c release after 5 Gy or 10 Gy. M30 demonstrated a dose-dependent increase in apoptotic index for a given increase in single-dose radiotherapy. There was a significant increase in apoptotic index between 1×2 Gy and 5×2 Gy. Conclusion M30 is a superior method compared with soluble markers in detecting low-dose radiation-induced cell death. This microfluidic technique can be used to assess radiation-induced cell death in HNSCC and therefore has the potential to be used to predict radiation response.
This communication reports for the first time the charging of a commercially available mobile phone, using Microbial Fuel Cells (MFCs) fed with real neat urine. The membrane-less MFCs were made out of ceramic material and employed plain carbon based electrodes.
The presence of regulatory T (Treg) cells is thought to be an important mechanism by which head and neck squamous cell carcinoma (HNSCC) successfully evades the immune system. Using multicolour flow cytometry, the frequency and functional capacity of two CD4(+) CD127(low/-) Treg cell populations, separated on the basis of different levels of CD25 expression (CD25(inter) and CD25(high) ), from the peripheral circulation of newly presenting HNSCC patients were assessed with regard to clinicopathological features and healthy controls. The frequency of circulating Treg cells was similar between HNSCC patients and healthy controls, and for patients with HNSCC developing from different subsites (laryngeal compared with oropharyngeal). However, patients with advanced stage tumours and those with nodal involvement had significantly elevated levels of CD4(+) CD25(high) CD127(low/-) Treg cells compared with patients who had early stage tumours (P = 0·03) and those without nodal involvement (P = 0·03), respectively. CD4(+) CD25(high) CD127(low/-) Treg cells from the entire HNSCC patient cohort and from patients whose tumours had metastasized to the lymph nodes were also shown to suppress the proliferation of effector T cells significantly more, compared with those from healthy controls (P = 0·04) or patients with no nodal involvement (P = 0·04). Additionally, CD4(+) CD25(inter) CD127(low/-) Treg cells consistently induced greater suppressive activity than CD4(+) CD25(high) CD127(low/-) Treg cells on the proliferation of the effector T-cell populations (CD4(+) CD25(-) CD127(-/+) and CD4(+) CD25(+) CD127(+) ). Peripheral Treg cells, identified by the CD127(low/-) phenotype, have been shown to be influenced by a patients tumour stage and/or nodal status in HNSCC; suggesting a role in tumour progression that could be manipulated by future immunotherapy.
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.
The levels of compounds in exhaled mouth air do not necessarily reflect levels in the systemic circulation as bacteria can bio-transform substrates to produce compounds within the mouth. This should be of concern to researchers measuring breath volatiles with the aim of diagnosing systemic or metabolic conditions as very little is known about the origin of many compounds detected on the breath. This pilot study investigated the production of volatile compounds by bacterial communities present within the mouth. Solid-phase micro-extraction was used to extract volatiles from the headspace gas of two Gram-positive and two Gram-negative bacterial cultures known to be present within the mouth and from tongue biofilm microflora. Analyses were undertaken using gas chromatography mass spectrometry. Between 64 and 82 volatile compounds were detected from sampling the headspace gas above each of the cultures. Gram-negative anaerobes were found to produce more volatile sulfur compounds (VSCs) and amines. Solobacterium moorei, a Gram-positive species was however found to produce higher levels of acids, hydrocarbons, alcohols and aldehydes than the other species studied. Tongue-scrape biofilm systems at lower pH gave more hydrocarbons, ketones and fatty acids whilst at higher pH more alcohols, aldehydes, VSCs and amines were detected in the headspace. The results show that a number of compounds detected in mouth breath are produced by anaerobic bacteria in tongue biofilms. Thus, the contribution of volatiles from oral anaerobes cannot be ignored and more research is required to identify the major source of breath compounds as this will help determine their clinical significance as indicators of systemic disease or metabolic disorders in the body.
Mutation of the p53 gene is a key event in the carcinogenesis of many different types of tumours. These can occur throughout the length of the p53 gene. Anti-p53 auto-antibodies are commonly produced in response to these p53 mutations. This review firstly describes the various mechanisms of p53 dysfunction and their association with subsequent carcinogenesis. Following this, the mechanisms of induction of anti-p53 auto-antibody production are shown, with various hypotheses for the discrepancies between the presence of p53 mutation and the presence/absence of anti-p53 auto-antibodies. A systematic review was performed with a descriptive summary of key findings of each anti-p53 auto-antibody study in all cancers published in the last 30 years. Using this, the cumulative frequency of anti-p53 auto-antibody in each cancer type is calculated and then compared with the incidence of p53 mutation in each cancer to provide the largest sample calculation and correlation between mutation and anti-p53 auto-antibody published to date. Finally, the review focuses on the data of anti-p53 auto-antibody in colorectal cancer studies, and discusses future strategies including the potentially promising role using anti-p53 auto-antibody presence in screening and surveillance.
The properties of earthenware and terracotta were investigated in terms of structural integrity and ion conductivity, in two microbial fuel cell (MFC) designs. Parameters such as wall thickness (4, 8, 18 mm), porosity and cathode hydration were analysed. During the early stages of operation (2 weeks), the more porous earthenware lost anolyte quickly and was unstable between feeding compared to terracotta. Three weeks later MFCs of all thicknesses were more stable and could sustain longer periods of power production without maintenance. In all cases, the denser terracotta produced higher open circuit voltage; however, earthenware the more porous and less iron-rich of the two, proved to be the better material for power production, to the extent that the thickest wall (18 mm) MFC produced 15 % higher power than the thinnest wall (4 mm) terracotta. After 6 weeks of operation, the influence of wall thickness was less exaggerated and power output was comparable between the 4 and 8 mm ceramic membranes. Cylindrical earthenware MFCs produced significantly higher current (75 %) and power (33 %) than terracotta MFCs. A continuous dripping mode of cathode hydration produced threefold higher power than when MFCs were submerged in water, perhaps because of a short-circuiting effect through the material. This shows a significant improvement in terms of biosystems engineering, since a previously high-maintenance half-cell, is now shown to be virtually self-sufficient.
Six continuous-flow Microbial Fuel Cells (MFCs) configured as a vertical cascade and tested under different electrical connections are presented. When in parallel, stable operation and higher power and current densities than individual MFCs were observed, despite substrate imbalances. The cascading dynamic allowed for a cumulative COD reduction of >95% in approximately 5.7h, equivalent to 7.97 kg COD m(-3) d(-1). Under a series configuration, the stack exhibited considerable losses until correct fluidic/electrical insulation of the units was applied, upon which the stack also exhibited superior performance. In both electrical configurations, the 6 MFC system was systematically starved for up to 15 d, with no significant performance degradation. The results from the 14-month trials, demonstrate that cascade-stacking of small units can result in enhanced electricity production (vs single large units) and treatment rates without using expensive catalysts. It is also demonstrated that substrate imbalances and starvation do not necessarily result in cell-voltage reversal.
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.
Microbial fuel cells (MFCs) generate electricity from waste but to date the technologys development and scale-up has been held-up by the need to incorporate expensive materials. A costly but vital component is the ion exchange membrane (IEM) which conducts protons between the anode and cathode electrodes. The current study compares natural rubber as an alternative material to two commercially available IEMs. Initially, the material proved impermeable to protons, but gradually a working voltage was generated that improved with time. After 6 months, MFCs with natural rubber membrane outperformed those with anion exchange membrane (AEM) but cation exchange membrane (CEM) produced 109 % higher power and 16 % higher current. After 11 months, polarisation experiments showed a decline in performance for both commercially available membranes while natural rubber continued to improve and generated 12 % higher power and 54 % higher current than CEM MFC. Scanning electron microscope images revealed distinct structural changes and the formation of micropores in natural latex samples that had been employed as IEM for 9 months. It is proposed that the channels and micropores formed as a result of biodegradation were providing pathways for proton transfer, reflected by the steady increase in power generation over time. These improvements may also be aided by the establishment of biofilms that, in contrast, caused declining performance in the CEM. The research demonstrates for the first time that the biodegradation of a ubiquitous waste material operating as IEM can benefit MFC performance while also improving the reactors lifetime compared to commercially available membranes.
This study reports for the first time on the development of a self-sustainable microbial fuel cell stack capable of self-maintenance (feeding, hydration, sensing & reporting). Furthermore, the stack system is producing excess energy, which can be used for improved functionality. The self-maintenance is performed by the stack powering single and multi-channel peristaltic pumps.
The nature of the tumour microenvironment immune response in head and neck cancer patients has an important role in tumour development and metastasis, but it is unknown if this differs between cancer subsites or whether it is related to the peripheral immune response.
In this modern era of multi-modality treatment there is increasing interest in the possibility of avoiding radical surgery in complete responders after neo-adjuvant long-course chemoradiotherapy (LCPRT). In this article, we present a systematic review of such treatments and discuss their therapeutic applicability for the future.
The aim of this study was to determine the effect of HNSCC tumour treatment on systemic Th1 and Th2 cytokine levels and investigate correlations with clinicopathological parameters. IL2, IL4, IL5, IL6, IL8, IL10, IL13, GMCSF, IFN? and TNF? were measured in the serum of 101 newly-presenting HNSCC patients (9 oral cavity, 27 oropharynx, 57 laryngopharynx, 1 sinonasal, 1 parotid and 6 unknown), prior to and following treatment, using a Quantibody(®) array based multiplex sandwich ELISA (Raybiotech). Data were analysed with respect to T stage, nodal status, age and sex of the patient as well as time between collection of pre- and post-treatment serum. A significant decrease in the levels of the Th2 cytokines IL4, IL5, IL6 and IL10 and the Th1 cytokines IL2 and IL8 was observed between the pre- and post-treatment serum samples. IL13 and TNF? were significantly higher in early stage (T1/T2) tumours compared with late stage (T3/T4) and this trend was maintained for nodal involvement. IL4 was higher in node positive patients compared with node negative, whereas the converse was true for IL2; IL4 was also higher in younger patients compared with the older age group. These results suggest that removal of HNSCC tumours from patients results in reduced circulating Th2 cytokines without a concurrent increase in Th1 cytokines, indicative of a partial rebalance of the Th1/Th2 system following treatment. Furthermore the cytokine profile may be influenced by the size and nodal involvement of the tumour.
This communication reports for the first time the direct utilisation of urine in MFCs for the production of electricity. Different conversion efficiencies were recorded, depending on the amount treated. Elements such as N, P, K can be locked into new biomass, thus removed from solution, resulting in recycling without environmental pollution.
Advanced pancreatic cancer is associated with a high risk of patients developing venous thromboembolism. This increased risk is thought to be tumour-driven and associated with tissue factor (TF) and microparticles. The aim of this study was to investigate the role of TF and phospholipid expression in the procoagulant properties of pancreatic cell lines and microparticles. Pancreatic cancer cell lines (MIA-PaCa-2, ASPC-1 and CFPAC-1) were assessed for expression of TF and microparticle release. Procoagulant potential was determined by a prothrombin time assay. Cell surface expression of TF was highest in CFPAC-1, with low expression on ASPC-1 and little/no expression on MIA-PaCa-2. Clotting time (CT) was cell number and TF-dependent (P?0.001). Blocking of TF resulted in slower CT for CFPAC-1 and ASPC1 and prevented clotting in MIA-PaCa-2. Microparticles were shown to be procoagulant and the majority of procoagulant potential could be removed by passing cell-free media through a 0.1 ?m filter. A dose-dependent CT was observed in both ASPC-1 and CFPAC-1 cell-free media. Furthermore, addition of duramycin prevented microparticle-supported coagulation. The data presented suggest a key role for cell and microparticle surface-expressed TF and phospholipids in coagulation and highlight duramycin-mediated disruption of clotting.
IL10, but not IL12 or T regulatory cells in the circulation of newly presenting, pre-treatment head and neck squamous cell carcinoma (HNSCC) patients, has been shown previously to be related to survival over a mean follow-up period of 15 months. Here, we followed the same patients for a longer period to determine whether these associations change. Pre- and post-treatment serum IL10/IL12 and circulating T regs were measured using ELISA and flow cytometry respectively and were correlated with survival after a 33 month average follow-up in a cohort of newly presenting HNSCC patients (n=107), with cancers of the hypopharynx (n=16), larynx (n=36), oral cavity (n=21), oropharynx (n=25), sinonasal (n=4) or unknown origin (n=5). Although the mean survival time of patients with detectable levels of IL10 pre-treatment was lower (40.6 months) than that of those without detectable levels of IL10 (45.6 months), the difference was no longer significant, in contrast to earlier follow-up data. In conclusion, although serum levels of IL10 may be a prognostic indicator for HNSCC patients over the short-term, they become less significant as follow-up time increases.
Current in vitro methodologies for the culture and analysis of liver specific responses lack the sophistication of in vivo dynamics. In this work, a microfluidic based experimental methodology has been utilized to reproduce a biomimetic microenvironment in which pseudo in vivo liver tissue studies can be carried out under in vitro conditions. This innovative technique, which exploits the inherent advantages of microfluidic technology, has been utilised to study the viability and functionality of explant liver tissue over four days in the presence of varying concentrations of ethanol. Concentrations of ethanol as low as 20 mM have produced a decrease in WST-1 metabolism, a marker of mitochondrial activity, and an increase lactose dehydrogenase release, reflecting cell death, in the explant samples; these effects increase with higher ethanol concentrations. A concomitant decrease in albumin and urea synthesis was also observed. We believe the proposed methodology is widely applicable and is clearly of relevance to biological and clinical research including drug development and toxicity, as well as enabling better fundamental understanding of tissue/cell processes.
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.
The expression of angiogenesis-related proteins was determined in laryngeal tumour tissue, associated tumour involved lymph nodes, apparently normal mucosa and control tissue and were related to tumour stage. Both laryngeal tumour tissue and associated metastatic nodes were obtained from seven patients undergoing surgical resection; in four cases apparently normal mucosa was also dissected from the tumour specimen margins. Control uvula mucosa was obtained from five healthy volunteers undergoing uvulopalatopharyngoplasty. The relative expression of 55 angiogenesis-related proteins was determined in tissue lysates using a Proteome Profiler human angiogenesis array kit. The level of 32/55 angiogenesis-related proteins was higher in tumour tissue compared with controls. Furthermore, in these tumour biopsies higher levels of proteins were associated with increasing tumour stage. A similar trend was seen for 29/32 of these proteins in the nodal tissue. In T4 stage tumour tissue samples, 29/55 angiogenensis-related proteins were more highly expressed compared with the adjacent normal mucosa from the same patient, and this decreased to 8 proteins in tumour tissue from the T1 stage patients. In contrast, the expression of 23 angiogenesis-related proteins in metastatic lymph node tissue from T4 stage patients was lower compared with that found in the normal mucosa adjacent to the tumour. In conclusion, this study has identified a number of factors involved in angiogenesis that are likely to contribute to the growth and metastasis of laryngeal tumours. Furthermore, a number of factors were also substantially altered in metastatic deposits compared with the primary tumour mass or adjacent normal tissue. This study requires confirmatory analysis of the selected key factors in a larger cohort of patients.
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.
Laboratory evidence of aberrant coagulation is found in the majority of patients with advanced pancreatic cancer and a clinical consequence of this is the high incidence and prevalence of vascular thromboembolic events. Other sequelae are hypothesized to be the facilitation and acceleration of mechanisms that define the malignant phenotype, such as invasion, trafficking and anchoring, establishing the metastatic niche and inducing angiogenesis. We review the in vitro and preclinical evidence that supports the role of the coagulation apparatus in the metastatic process of pancreatic cancer, with a particular emphasis on interaction of this pathway with clinically-targeted growth factor receptor pathways. Links between hemostasis, angiogenesis and epidermal growth factor pathways and their significance as therapeutic targets are considered.
A method for assessing the performance of microbial fuel cells (MFCs) is the polarisation sweep where different external resistances are applied at set intervals (sample rates). The resulting power curves often exhibit an overshoot where both power and current decrease concomitantly. To investigate these phenomena, small-scale (1 mL volume) MFCs operated in continuous flow were subjected to polarisation sweeps under various conditions. At shorter sample rates the overshoot was more exaggerated and power generation was overestimated; sampling at 30 s produced 23% higher maximum power than at 3 min. MFCs with an immature anodic biofilm (5 days) exhibited a double overshoot effect, which disappeared after a sufficient adjustment period (5 weeks). Mature MFCs were subject to overshoot when the anode was fed weak (1 mM acetate) feedstock with low conductivity (<100 ?S) but not when fed with a higher concentration (20 mM acetate) feedstock with high conductivity (>1500 ?S). MFCs developed in a pH neutral environment produced overshoot after the anode had been exposed to acidic (pH 3) conditions for 24 h. In contrast, changes to the cathode both in terms of pH and varying catholyte conductivity, although affecting power output did not result in overshoot suggesting that this is an anodic phenomenon.
The continuous flow focussing and manipulation of particles and cells are important factors in microfluidic applications for performing accurate and reproducible procedures downstream. Many particle focussing methods require complex setups or channel designs that can limit the process and its applications. Here, we present diamagnetic repulsion as a simple means of focussing objects in continuous flow, based only on their intrinsic properties without the requirement of any label. Diamagnetic polystyrene particles were suspended in a paramagnetic medium and pumped through a capillary between a pair of permanent magnets, whereupon the particles were repelled by each magnet into the central axis of the capillary, thus achieving focussing. By investigating this effect, we found that the focussing was greatly enhanced with (i) increased magnetic susceptibility of the medium, (ii) reduced flow rate of the suspension, (iii) increased particle size, and (iv) increased residence time in the magnetic field. Furthermore, we applied diamagnetic repulsion to the flow focussing of living, label-free HaCaT cells.
The increasing prevalence of multi-drug resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), necessitates development of alternative modes of bacterial targeting which are not hindered by antibiotic resistance and minimise collateral damage. To achieve this, the FliTrx™ bacterially-displayed peptide library was panned against MRSA and randomly selected clones (n = 20) were DNA sequenced. One selected peptide was synthesised as both cyclic and linear constructs. Binding of the cyclic construct was observed by flow cytometry against isolates of MRSA whilst the linear construct showed low affinity. Low reactivity was observed with other Staphylococcal sp., gram-negative bacteria and human keratinocytes. The selected peptide was also cloned in-frame, within the thioredoxin gene into the pPROTet.E 6xHN vector for protein expression. A porphyrin photosensitiser (5-(4-isothiocyanatophenyl)-10,15,20-tris(4-N-methylpyridiniumyl)porphyrin trichloride) was conjugated to the recombinant protein and the in vitro cytotoxic effect of the resulting bioconjugate was determined against MRSA and other non-specific bacterial and mammalian cell lines. Photoantimicrobial chemotherapy (PACT) using the bioconjugate showed a 66% reduction in MRSA growth in comparison with non-irradiated cells. This work demonstrates the potential to isolate peptides with binding specificity against MRSA that can be used for targeted PACT, providing an effective alternative to antibody targeting.
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.
Two cationic porphyrins bearing an isothiocyanate group for conjugation to monocolonal antibodies have been synthesized. The two porphyrins conjugated efficiently to three monoclonal antibodies (anti-CD104, anti-CD146 and anti-CD326), which recognize antigens commonly over-expressed on a range of tumour cells. In vitro, all conjugates retained the phototoxicity of the porphyrin and the immunoreactivity of the antibody. Mechanistic studies showed that conjugates formed from the mono- and tri-cationic porphyrin and anti-CD104 antibody mediated apoptosis following irradiation with non-thermal red light of 630 ± 15 nm wavelength. In vivo antibody conjugates caused suppression of human LoVo tumour growth in immunodeficient NIH III mice, similar to the commercial photodynamic therapy (PDT) agent Photofrin, but at administered photosensitizer doses that were more than two orders of magnitude lower. Positron emission tomography (PET) following PDT showed a large, early increase in uptake of (18) fluorodeoxyglucose (FDG) by tumours treated with the anti-CD104 conjugates. This effect was not observed with Photofrin or with conjugates formed from the same photosensitizers conjugated to an irrelevant antibody.
Numerous chemotherapeutic regimens exist for the treatment of symptomatic or progressive chronic lymphocytic leukaemia (CLL). However, once the disease becomes refractory to nucleoside-based therapy the prognosis is poor. In this study we investigated the cytotoxicity of thalidomide in combination with dexamethasone, fludarabine and cyclophosphamide. Cells from a cohort of 25 CLL patients were incubated for 72 h with each of these three agents, at 3 concentrations, both with and without thalidomide. Cell viability was assessed using the Annexin V:FITC assay. Fludarabine was highly toxic to the cells, producing very high levels of cell death; however, thalidomide did not increase this effect. Cyclophosphamide combined with thalidomide showed a small, non-significant improvement in toxicity compared with monotherapy. Median cell death for 5 µM dexamethasone monotherapy and for combination with thalidomide was 15% [interquartile range (IQR) 0-38%] and 17% (IQR 0-54%), respectively (Wilcoxon Signed Rank analysis, p=0.034). Cell death for 10 µM dexamethasone monotherapy was 15% (IQR 0-45%) and 16% (IQR 0-62%) in combination with thalidomide (Wilcoxon Signed Rank analysis, p=0.035). At the highest doses tested 11 of 25 cases displayed an enhancement of cyclophosphamide-mediated cytotoxicity, and 14 of 25 cases showed enhanced dexamethasone-mediated cytotoxicity in the presence of thalidomide. Some CLL cells in which dexamethasone-mediated killing was enhanced were derived from patients with poor prognostic markers, including p53 mutations and unmutated IgVH genes. In summary, thalidomide enhances cyclophosphamide- and dexamethasone-mediated cytotoxicity of CLL cells in vitro in a proportion of cases.
Volatile compounds (VCs) are produced by all microorganisms as part of their normal metabolism. The aim of this study was to determine whether bacterial VC profiles could be used to discriminate between selected bacterial species and strains in vitro. Selected Ion Flow Tube Mass Spectrometry (SIFT-MS) was used to quantify the concentration of 23 microbial VCs within the head-space of various bacterial monocultures, during both the logarithmic and stationary growth phases. In comparison with existing techniques, SIFT-MS enables quantitative, high throughput, real-time head-space analysis to be performed, without need for sample preparation. The results show that most VCs were produced by >1 bacterial species or strain, and some were produced by all strains tested. Multivariate analysis using similarity matrices, cluster analysis and multidimensional scaling (MDS) was used to determine whether there was a characteristic VC profile at either the species or strain level. Significant discrimination of all bacterial species and strains was achieved by analysing the VC profiles, and the relative similarity of VC profiles could be differentiated in 2 or 3 dimensional space. This study has shown that there are significant differences in the volatile profiles obtained from various bacterial monocultures grown in vitro, and that the analysis techniques herein employed have the potential to differentiate samples at the strain level.
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.
Microbial fuel cells (MFCs) can treat wastewater but individually are thermodynamically restricted. Scale-up might, therefore, require a plurality of units operating in a stack which could introduce losses simply through fluidic connections. Experiments were performed on two hydraulically joined MFCs (20 cm apart) where feedstock flowed first through the upstream unit (MFC(up)) and into the downstream unit (MFC(down)) to explore the interactive effect of electrical load connection, influent make-up and flow-rate on electrical outputs. This set-up was also used to investigate how calculating total internal resistance based on a dynamic open circuit voltage (OCV) might differ from using the starting OCV. When fed a highly conductive feedstock (~4,800 ?S) MFC(down) dropped approximately 180 mV as progressively heavier loads were applied to MFC(up) (independent of flow-rate) due to electron leakages through the medium. The conductivities of plain acetate solutions (5 and 20 mM) were insufficient to induce losses in MFC(down) even when MFC(up) was operating at high current densities. However, at the highest flow-rate (240 mL/h) MFC(down) dropped by approximately 100 mV when using 5 and 220 mV using 20 mM acetate. When the distance between MFCs was reduced by 5 cm, voltage drops were apparent even at lower flow-rates, (30 mL/h decreased the voltage by 115 mV when using 20 mM acetate). Shear flow-rates can introduce dissolved oxygen and turbulence all capable of affecting the anodic biofilm and redox conditions. Calculating total internal resistance using a dynamic OCV produced a more stable curve over time compared to that based on the starting constant OCV.
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.
The aim of the present study was to assess the role of tissue factor and serum-induced cell invasion in patients with advanced pancreatic cancer (APC). A cohort of 39 patients with APC, without thrombosis, receiving chemotherapy, were entered in a randomized controlled trial (ISRCTN = 76464767) of thromboprevention with weight-adjusted dalteparin (WAD). A total of 19 patients received WAD, the remaining 20 acting as a control group. Serum from baseline and week 8 was analysed for circulating-tissue factor antigen using ELISA. Circulating-tissue factor antigen rose from 324 pg/ml, [interquartile range (IQR) 282-347 pg/ml] to 356 pg/ml, (IQR 319-431 pg/ml) in controls (C), and decreased in the dalteparin-treated group (D) from 336 pg/ml (IQR 281-346 pg/ml) to 303 pg/ml (IQR 274-339 pg/ml). The difference in median percentage change between D and C was statistically significant [-4.0 (D) vs. 4.7 (C); P = 0.005, n = 39]. Serum-induced cellular invasion of MIA-Paca-2 cells in response to patient serum was studied using a Boyden chamber assay in 30 patients (14 WAD and 16 C). The median percentage change between C and D was significant [+54.9 (C) vs. -21.9 (D) P = 0.025, n = 30]. There was a weak correlation between BB-tissue factor reduction and cellular invasion reduction (Spearman) [0.384 (P = 0.037, n = 30)]. APC patients treated with WAD have lower tissue factor antigen levels and attenuated induction of cellular invasion in their blood. These assays may provide useful markers to guide appropriate dalteparin (and other low-molecular weight heparin) dosing schedules to optimize anticancer effects of dalteparin in APC.
The pH of the tongue biofilm is likely to influence microbial composition and ecology with consequent effects on the metabolic activities and generation of volatile sulfur compounds (VSC) and other malodour gasses. The aim of this study was to identify the effects of pH on the development of biofilms and hydrogen sulfide production using an in vitro tongue-derived biofilm model. Community level physiological profiling (CLPP) was employed to examine the influence of pH on the collective metabolic fingerprint of each tongue-derived biofilm. A sorbarod perfusion system (n = 6 sorbarods) was inoculated from a single suspension of tongue scrape sample and mixed community tongue-derived biofilms were grown at pH 5.5, 6.0, 6.5, 7.0 7.5 and 8.0. Biofilms were perfused with medium for 120 h and gas phase samples (n = 4 per biofilm) removed and analysed with a portable sulfide gas chromatograph before being sacrificed into 10 ml sterile PBS-diluent and cells suspended by vortex mixing. Further ten-fold dilutions were made (down to 10(-7)) and dilutions plated out onto selective (fastidious anaerobic agar (FAA) + 0.0025% vancomycin) and non-selective (FAA) media for enumeration of strict and facultative anaerobes respectively. Biofilm suspensions were also mixed with Biolog inoculation fluid and distributed into 96 wells of Biolog AN plates for CLPP. Tongue biofilms developed at pH 7.5 produced significant (p < 0.05) concentrations of H(2)S (?52.2 ± SEM 5.6 µg H(2)S per ml biofilm gas phase) followed by tongue biofilm developed at pH 7.0 and 8.0 (?43.2 ± SEM 3.5 and ? 39.6 ± SEM 7.3 µg H(2)S per ml biofilm gas phase respectively). Tongue biofilm developed at pH 6.0 and 6.5 produced approximately 21.5 ± SEM 2.3 and 37.1 ± SEM 1.7 µg H(2)S per ml biofilm gas phase respectively and tongue biofilm developed at pH 5.5 produced approximately 0.19 ± SEM 0.09 µg H(2)S per ml biofilm gas phase. Highest numbers of strict and facultative anaerobes were recovered from biofilms at pH 6.5 (1.10 × 10(12) and 2.07 × 10(12) cfu ml(-1) respectively), with a reduced number recovered from pH values above and below this range. CLPP and similarity index revealed biofilms at pH 6.5 and 7.0 most similar (S(j) = 78%) and most diverse in terms of metabolic activity. The biofilm at pH 5.5 was the least related to all others and least diverse. The sorbarod perfusion system, in conjunction with H(2)S analysis and CLPP, enables some of the physiological and ecological effects of pH at a local level within the biofilm on H(2)S production to be identified.
To process large volumes of wastewater, microbial fuel cells (MFCs) would require anodophilic bacteria preferably operating at high flow-rates. The effect of flow-rate on different microbial consortia was examined during anodic biofilm development, using inocula designed to enrich either aerobes/facultative species or anaerobes. All MFCs underperformed at high flow-rates in the early stages, however, the aerobic type - following anodic biofilm development - subsequently exhibited more marked improvement. Scanning electron microscopy showed some variation in biofilm formation where clumpy growth was associated with lower power. Over time both power and internal resistance increased for the low flow-rates perhaps explained by an evolving microflora that consequently changed redox potential. An overshoot was observed in power curves, which was attributed to increased internal resistance due to ionic depletion and/or microbial exhaustion. To the best of the authors knowledge this is the first time that such phenomena are explained from the internal resistance perspective.
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.
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.
Serum vascular endothelial growth factor-A has been associated with stage of disease and prognosis in colorectal cancer. In this study, the clinical usefulness of preoperative serum vascular endothelial growth factor-A concentrations in the long-term follow-up of colorectal cancer patients was evaluated.
Vascular endothelial growth factor-C (VEGF-C) is one of the most potent lymphangiogenic members of the VEGF family that has been associated with lymph node metastasis and poor prognosis in patients with colorectal cancer (CRC). In this study, we evaluated the relationship of preoperative serum VEGF-C (sVEGF-C) and survival in CRC patients.
Three Microbial Fuel Cells (MFCs) were fluidically connected in series, with a single feed-line going into the 1st column through the 2nd column and finally as a single outflow coming from the 3rd column. Provision was also made for re-circulation in a loop (the outflow from the 3rd column becoming the feed-line into the 1st column) in order to extend the hydraulic retention time (HRT) on treatment of landfill leachate. The effect of increasing the electrode surface area was also studied whilst the columns were (fluidically) connected in series. An increase in the electrode surface area from 360 to 1080 cm(2) increased the power output by 118% for C2, 151% for C3 and 264% for C1. COD and BOD(5) removal efficiencies also increased by 137% for C1, 279% for C2 and 182% for C3 and 63% for C1, 161% for C2 and 159% for C3, respectively. The system when configured into a loop was able to remove 79% of COD and 82% of BOD(5) after 4 days. These high levels of removal efficiency demonstrate the MFC systems ability to treat leachate with the added benefit of generating energy.
An integrated gel supported micro-fluidic system is reported, in which PCR products can be efficiently injected into a capillary electrophoresis device. The gel supported system is designed to provide greater stability to reagents during long periods of dormancy, enabling the mass production of one use chips encapsulating all required reagents at the time of manufacturing. This simultaneously diminishes the risk of sample contamination, and reduces the amount of external hardware required for auxiliary flow control, thus increasing the potential for portability. After PCR amplification was performed in a polysaccharide gel matrix, the PCR product was injected into the separation gel polymer matrix by executing a capillary-based electro-kinetic pinched injection across a gel-to-gel interface. The gel-to-gel system delivered a precise and accurate plug into the separation polymer, which offered more stable electro-kinetic control of the sample compared to solution based methodology even when bubbles were present in the system. Suitable voltage control was proven to provide a repeatable electro-kinetic injection of PCR product sufficient for an on-chip separation of multiple loci by capillary electrophoresis.
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.
A new copper(II) containing bis-macrocyclic CXCR4 chemokine receptor antagonist is shown to have improved binding properties to the receptor protein in comparison to the drug AMD3100 (Plerixafor, Mozobil). The interaction of the metallodrug has been optimized by using ultrarigid chelator units that offer an equatorial site for coordination to the amino acid side chains of the protein. Binding competition assays with anti-CXCR4 antibodies show that the new compound stays bound longer and it has improved anti-HIV potency in vitro (EC(50) = 4.3 nM). X-ray structural studies using acetate as a model for carboxylate amino acid side chains indicate the nature of the coordination interaction.
Although malignant pleural mesothelioma is a rare tumour, its incidence is increasing. The prognosis remains very poor with an average survival of 10 months from diagnosis. The choice of chemotherapy regimens for mesothelioma patients is limited and new approaches are required. COX-2 inhibition induces apoptosis in a variety of tumour cell lines. The cytotoxic effect of conventional drugs may be enhanced by the addition of a COX-2 inhibitor. In order to identify possible new therapeutic approaches we aimed to determine whether the addition of COX-2 inhibitors would enhance the cytotoxic effect of chemotherapeutic agents in mesothelioma cell lines.
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.
Cancer is frequently complicated by venous thromboembolic events (VTE), which pose a significant health burden due to the associated high morbidity and mortality rates, yet the exact details of the pathophysiological mechanisms underlying their development are yet to be fully elucidated. Tissue factor (TF), the primary initiator of coagulation, is often overexpressed in malignancy and as such is a prime candidate in predicting the hypercoagulable state. Further exploration of this potential role has identified increases in the number of TF-expressing microparticles (MP) in the circulation of cancer patients, in particular in those known to have high incidences of thromboembolic complications. The risk of VTE in cancer is found to be further elevated by chemotherapy. Chemotherapy may, in eliciting cancer cell apoptosis, result in an increase in release of circulating procoagulant MP. We discuss a potential role of elevated tumour TF expression and increased circulating TF-positive MP in predicting VTE risk.
The aim of this work is to study the relationship between growth rate and electricity production in perfusion-electrode microbial fuel cells (MFCs), across a wide range of flow rates by co-measurement of electrical output and changes in population numbers by viable counts and optical density. The experiments hereby presented demonstrate, for the first time to the authors knowledge, that the anodic biofilm specific growth rate can be determined and controlled in common with other loose matrix perfusion systems. Feeding with nutrient-limiting conditions at a critical flow rate (50.8 mL h(-1)) resulted in the first experimental determination of maximum specific growth rate ?(max) (19.8 day(-1)) for Shewanella spp. MFC biofilms, which is considerably higher than those predicted or assumed via mathematical modelling. It is also shown that, under carbon-energy limiting conditions there is a strong direct relationship between growth rate and electrical power output, with ?(max) coinciding with maximum electrical power production.
The development of the microbial fuel cell (MFC) technology has seen an enormous growth over the last hundred years since its inception by Potter in 1911. The technology has reached a level of maturity that it is now considered to be a field in its own right with a growing scientific community. The highest level of activity has been recorded over the last decade and it is perhaps considered commonplace that MFCs are primarily suitable for stationary, passive wastewater treatment applications. Sceptics have certainly not considered MFCs as serious contenders in the race for developing renewable energy technologies. Yet this is the only type of alternative system that can convert organic waste-widely distributed around the globe-directly into electricity, and therefore, the only technology that will allow artificial agents to autonomously operate in a plethora of environments. This Minireview describes the history and current state-of-the-art regarding MFCs in robotics and their vital role in artificial symbiosis and autonomy. Furthermore, the article demonstrates how pursuing practical robotic applications can provide insights of the core MFC technology in general.
Microbial cultures and/or microbial associated diseases often have a characteristic smell. Volatile organic compounds (VOCs) are produced by all microorganisms as part of their normal metabolism. The types and classes of VOC produced is wide, including fatty acids and their derivatives (e.g. hydrocarbons, aliphatic alcohols and ketones), aromatic compounds, nitrogen containing compounds, and volatile sulfur compounds. A diversity of ecological niches exist in the human body which can support a polymicrobial community, with the exact VOC profile of a given anatomical site being dependent on that produced by both the host component and the microbial species present. The detection of VOCs is of interest to various disciplines, hence numerous analytical approaches have been developed to accurately characterize and measure VOCs in the laboratory, often from patient derived samples. Using these technological advancements it is evident that VOCs are indicative of both health and disease states. Many of these techniques are still largely confined to the research laboratory, but it is envisaged that in future bedside VOC profiling will enable rapid characterization of microbial associated disease, providing vital information to healthcare practitioners.
Heat shock protein 72 (Hsp72) is a highly inducible stress protein and molecular chaperone. Cancers have been shown to be associated with increased Hsp72 expression within the tumour itself and this may lead to resistance to apoptosis.
Duramycin is a polypeptide that binds specifically to phosphatidylethanolamine (PE) on cell surfaces with high affinity, and has been shown to disrupt tumour cell surface-based coagulation and exhibit weak antimicrobial activity. The aim of the present study was to characterize the effect of duramycin on tumour cell proliferation and viability. Duramycin was used to detect phosphatidylethanolamine expression on cell lines by flow cytometry. Cells were cultured in the presence of duramycin and proliferation and cell viability assessed. Electron microscopy and confocal microscopy were utilized to investigate cell membrane structure after duramycin treatment. Pancreatic tumour cells were shown to express phosphatidylethanolamine on their cell surfaces by specific labelling with duramycin. Phosphatidylethanolamine expression was generally increased in apoptotic cells and more so in necrotic cells. Cells cultured in the presence of duramycin showed increasing levels of apoptosis and ultimately necrosis with increasing duramycin concentrations, and cell proliferation was reduced in a duramycin dose-dependent manner between 0.125 and 12.5 ?mol/l. Tissue factor expression was also reduced when cells were cultured in the presence of duramycin. Cells imaged by electron microscopy were fragile, suggesting that membrane integrity was compromised by duramycin, although no obvious differences in membrane structure were observed by live cell confocal imaging. Duramycin induced apoptosis and exhibited antiproliferative and anticoagulant effects on pancreatic tumour cells, most probably by disrupting cell membrane structure and/or function.
Seven miniature microbial fuel cells (MFCs) were hydraulically linked in sequence and operated in continuous-flow (cascade). Power output and treatment efficiency were investigated using varying organic loads, flow-rates and electrical configurations. When fed synthetic wastewater low in organic load (1mM acetate) only the first MFC operated stably over a 72-h period. Acetate feedstock at 5mM was enough to sustain the first four MFCs, and 10mM acetate was sufficient to maintain all MFCs at stable power densities. COD was reduced from 69 to 25mg/L (64%, 1mM acetate), 319-34mg/L (90%, 5mM acetate) and 545-264mg/L (52%, 10mM acetate). Fluctuating flow-rates improved performance in downstream MFCs. When connected electrically in parallel, power output was two-fold and current production 10-fold higher than when connected in series. The results suggest cascades of MFCs could be employed to complement or improve biological trickling filters.
Microfluidics is an emerging multidisciplinary field that has the potential to provide solutions to many of the current challenges in managing head and neck squamous cell carcinoma (HNSCC). Treatment strategies for this disease are often complex and associated with significant morbidity and mortality; furthermore, prediction of response to treatment remains inaccurate. Microfluidic technology allows a small sample of tumor to be maintained alive ex vivo within a microenvironment that mimics the in vivo milieu; the response of this tumor biopsy to treatment regimens can subsequently be investigated. Microfluidics is the science and technology of systems that process or manipulate microliter to nanoliter volumes of fluids in purpose-built devices fabricated from glass or other biocompatible polymers. The technology also involves miniaturization and integration of various laboratory procedures into novel analysis devices. The purpose of this review is to provide an overview of microfluidic devices and discuss potential applications in HNSCC management.
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.
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