Professor Duncan Sharp

The exploitation of singlet oxygen generating compounds as a means of targeted therapies drives the need to develop methods for assessing the efficacy of such compounds and their capacity for generating the reactive oxygen species. Degradation of diphenylisobenzofuran by singlet oxygen is widely used as a spectroscopic probe but its application can be problematic. An alternative detection strategy exploiting the electrochemical monitoring of the quencher concentration, by square wave voltammetry, has been shown to provide a more sensitive and flexible option that could be used to address the increasing interest in photosensitizing materials

The prevalence of antibiotic resistance has increased during the last few years and is viewed as a growing problem which has fuelled copious amounts of research on the development of new antibacterial agents. Silver is well recognized as possessing antibacterial activity and so by harnessing these properties and incorporating silver nanoparticles (AgNPs) within microdevices possessing microfiuidic channels has much promise. Progress in developing these types of systems has been limited due to the technological difficulties involved in controlling the inclusion of these nanoparticles within the devices. This work provides an insight into a novel electrochemical interaction that can enable not only the localisation of silver within such structures but also enables the smart release of AgNPs.

With the recent increase in bacterial resistance to conventional antibiotics, the early-stage detection and control of infection has become imperative in the fight against opportunistic pathogens in healthcare. The traditional β-lactam wonder-drugs (e.g. penicillin and cephalosporins), are rendered inactive due to enzymatic hydrolysis by bacterial β-lactamase enzymes as a bacterial defence mechanism. However, this deactivation mechanism produces different responses in the two aforementioned drugs - with the cephalosporins showing a molecular rearrangement mechanism which could be utilised for prodrug delivery. This unique mechanism could mean that inactive forms of cephalosporin antibiotics, once used as chemotherapeutics in oncology, could once again be used in the fight against disease as sensors to detect and treat bacterial colonisation. Therefore, we hypothesize that cephalosporin-dye bandages might provide an effective method to visually detect, and subsequently control, the early stages of an infection using photoantimicrobial chemotherapy (PACT).

An interdisciplinary and engaging practical is detailed which offers great versatility in the study of a qualitative and quantitative metabolism of azo-dyes by Staphylococcus aureus. This practical has broad scope for adaptation in the number and depth of variables to allow a focused practical experiment or small research project. Azo-dyes are an important group of dyes characterised by the presence of an azo-bond within the molecule. Many bacteria are able to produce azoreductases - enzymes which cleave the azo-bond of such dyes, decolourising them. This visual representation of metabolism offers a simple and inexpensive method to study bacterial metabolism and the variables which may affect this. The practical experiment combines basic microbiological and biochemical techniques whereby broth cultures are incubated with an azo-dye, and dye degradation is photometrically monitored. Microbiological methods may also be employed to monitor the resulting microbial growth. Example results are shown from student data to highlight the effects of differing broth media and how metabolism and growth may change over a 24-hour incubation period. The performance, analysis and interpretation of the findings may prove beneficial to test a variety of skills and aptitudes within both secondary and tertiary biological education. © 2012 Society of Biology.

New technologies are essential for intelligent wound management and to provide tools that facilitate a greater understanding of wounds and healing physiology. pH is an important marker for many processes in the wound environment; it cannot be fully utilised due to the inherent lack of suitable technologies currently available. The development and proof-of-concept testing for an electrochemical system that exploits pad-printed carbon-uric acid composite electrodes is detailed. Uric acid is incorporated to act as a biologically-safe pH probe within in the sensor assembly that can be manipulated to offer a simple voltammetric response. The development of the composite sensors, the activation of the basal carbon, and the surface deposition of 1,2-diaminobenzene to prevent biofouling are detailed. The prototype sensing assembly is shown to enable the interference-free measurement of pH (and linear quantification of endogenous uric acid) even in the presence of high ascorbic acid concentrations. The experimental developments culminate in a standard deviation of 0.164 for 20 replicates performed in simulated wound fluid, and sensitive monitoring of pH across a wide analytical range (pH 4-10) in simulated wound fluid. These findings suggest that printed carbon-uric acid composites may offer a novel, cheap and reliable mechanism for simple pH measurements at wound surfaces, a potentially powerful tool with clinical utility for wound management and one that may enable a greater understanding of pH implications on wound physiology, and the effects of dressings and treatments.

With the recent increase in bacterial resistance to conventional antibiotics, the early-stage detection and control of infection has become imperative in the fight against opportunistic pathogens in healthcare. The traditional β-lactam wonder-drugs (e.g. penicillin and cephalosporins), are rendered inactive due to enzymatic hydrolysis by bacterial β-lactamase enzymes as a bacterial defence mechanism. However, this deactivation mechanism produces different responses in the two aforementioned drugs - with the cephalosporins showing a molecular rearrangement mechanism which could be utilised for prodrug delivery. This unique mechanism could mean that inactive forms of cephalosporin antibiotics, once used as chemotherapeutics in oncology, could once again be used in the fight against disease as sensors to detect and treat bacterial colonisation. Therefore, we hypothesize that cephalosporin-dye bandages might provide an effective method to visually detect, and subsequently control, the early stages of an infection using photoantimicrobial chemotherapy (PACT). © 2012 Elsevier Ltd.

The applicability of employing a carbon fibre mesh as an electrochemical sensing substructure for assessing urate transformations within wound exudates is evaluated. Prototype sensor assemblies have been designed and their response characteristics towards uric acid and other common physiological components are detailed. Modification of the carbon fibre sensor through surface anodization and the application of cellulose acetate permselective barriers have been shown to lead to optimized responses and much greater sensitivity (1440% increase) and specificity. These could enable the accurate periodic monitoring of uric acid in wound fluid. The performance characteristics of the composite sensors in whole blood, serum and blister fluid have been investigated. © 2008 The Japanese Biochemical Society.

Pyocyanin is produced by Ps. aeruginosa as a result of quorum sensing during wound colonisation increasing bacterial virulence and damaging host physiology, both of which contribute to an increased risk of infection. The use of carbon fibre tow as an electrochemical sensing matrix for assessing pyocyanin production is evaluated. Prototype sensor assemblies have been developed and response characteristics towards pyocyanin are detailed. The sensitive and linear quantification of pyocyanin is presented (r2 = 0.998) across the biomedically relevant concentration range (1–100 µM). Precise electrochemical measurements of pyocyanin by square wave voltammetry are established using carbon fibre assemblies (coefficient of variance = 1.2 and 1.4% for 10 and 50 µM pyocyanin, respectively). Further testing of the sensors in bacterial cultures shows the ability to monitor pyocyanin production by Ps. aeruginosa in agreement with the chloroform-acid/photometric method and in the presence of other bacterially derived pigments and metabolites. The proposed small and inexpensive sensor assembly is suggested for use in monitoring Ps. aeruginosa growth.

The combination of reverse iontophoresis and flame photometry provides an engaging analytical experiment that gives first-year undergraduate students a flavor of modern drug delivery and analyte extraction techniques while reinforcing core analytical concepts. The experiment provides a highly visual demonstration of the iontophoresis technique and engenders a more active participation in the analytical process. The construction of a safe and cost-effective iontophoresis system is described that has proven to be sufficiently robust for use within the undergraduate laboratory. © 2010 The American Chemical Society and Division of Chemical Education, Inc.

The applicability of employing a carbon fibre mesh as the sensing element within a “smart bandage” for assessing urate transformations within wound exudates is evaluated and a novel strategy for the detection of bacterial contamination presented. Prototype sensor assemblies have been designed and their response characteristics towards the periodic monitoring of uric acid within whole blood, serum, blister fluid and microbial culture has been evaluated. The rapid and selective metabolism of urate by Pseudomonas aeruginosa, the bacteria responsible for most adventitious wound infections, has been investigated. A preliminary evaluation of the efficacy of utilizing the microbial response to endogenous wound urate as means of detecting the onset of infection is presented.

The use of laser ablation as a method through which the simultaneous patterning and activation of carbon fibre electrodes can be achieved has been investigated. Laser-induced exfoliation of the carbon fibre substrates was observed leading to creation of edge plane sites that enhance electrode performance. The laser-mediated surface modification is compared to that attainable using conventional surface anodisation using electrochemical means. © 2008 Elsevier Ltd. All rights reserved.

The exploitation of the natural product plumbagin (5-hydroxy-2-methyl-1,4- naphthoquinone) to produce polymer films capable of serving as controllable source of reactive oxygen species is assessed. The efficacy of the film to catalyse the reduction of oxygen to yield reactive oxygen species has been evaluated through using ascorbate and glutathione redox probes. © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2010.

Carbon based materials have a wel-founded history in electroanalytical science, and over the previous 55 years have offered many developments in terms of form, properties and modifications that can modulate the electrochemical properties. This review explores some of the more recent developments in carbon materials focussing on the versatility of graphitic carbon, its modification and a summative overview of the use of printing methodologies to fabricate electrodes and sensors based on modified carbon inks. A range of printed carbon electrodes and biosensors are discussed, based on the incorporation of electrocatalyst, mediator, modifier or enzyme, directly into the ink, to offer sensitive detection mechanisms. Future possibilities and strategies are discussed to explore ways in which printed carbon composites may be benefit electrochemistry and provide an emerging basis for cost effective detection technologies that are easily translated from research into commercial use.

Microwave irradiation was applied to a sequence of condensation reactions from readily available 9-chloroacridines to provide a range of novel acridine–isoalloxazine conjugates. The combination of these two moieties, both of biological interest, was achieved by a chromatography-free route.

Abstract

Intermediary metabolism can be a complex area to study due to the inherent modularity of the catabolic biochemical processes. This article outlines a novel, cost‐effective, and universally applicable teaching activity to enhance students understanding of the inter‐relationship between the key processes of intermediary metabolism. A simple origami model, the “Biochemist‐Tree” has been developed as a learning activity for intermediary metabolism, and its implementation was assessed. This modular origami model involves folding individual layers of the model, which must then be constructed into a “tree” with the metabolic process flowing correctly from macromolecules to ATP production from the electron transport chain/oxidative phosphorylation. Forty‐eight students were provided with the activity as a revision tutorial exercise toward the end of a module on the introduction to biochemistry. The findings from this study show that the students found the activity to be interesting (>75%), scientifically useful (95%), and would recommend other students (85%) using this for their revision of catabolic metabolism. The novel nature of the exercise meant that it was engaging for the students and one that they also felt improved the understanding of the integration of the metabolic processes included. Further to being a one‐off test of student understanding, the inclusion of key molecular structures and pathway steps helps to further embed these as a reference sources for revision. © 2013 by The International Union of Biochemistry and Molecular Biology, 41(5):309–314, 2013

This proof-of-concept study explores the novel use of carbon-based electrodes for the electrochemical generation of hypochlorite, and compares the antimicrobial efficacy against commercial hypochlorite solution. Antimicrobial concentrations of hypochlorite were generated using pad-printed carbon and carbon-fibre electrodes, yielding up to 0.027% hypochlorite in 60 minutes, and 0.1% hypochlorite in 15 minutes, respectively, in a non-divided assembly. The minimum inhibitory concentration (MIC) of the electrogenerated hypochlorite produced using carbon fibre electrodes was established for four medically important bacteria (Pseudomonas aeruginosa and Staphylococcus aureus ~0.025%, Escherichia coli and Enterococcus faecalis ~0.012%) and found to be in agreement with those determined using commercial hypochlorite solution. Therefore, carbon-based electrodes, particularly carbon fibre, have proven effective for the generation of antimicrobial concentrations of hypochlorite. The similarity of the MIC values to commercial hypochlorite solutions suggests that the antimicrobial efficacy is derived from the quantified hypochlorite generated and not due to marked co-generation of reactive oxygen species, as identified for other assemblies. As such, the application of carbon electrodes may be suitable for the local production of hypochlorite for healthcare antisepsis. This article is protected by copyright. All rights reserved.

Pyocyanin, a toxin produced by Pseudomonas aeruginosa, offers potential as a biomarker for the indirect detection of this bacterium of major importance for infections in burns, woundcare and cystic fibrosis. Pad-printed carbon electrodes are herein explored using square wave voltammetry to detect pyocyanin in a range of buffered and biological media. Third-order polynomial baseline fitting was explored to enhance the analytical sensitivity and extend the linear range to submicromolar concentrations. These modelling baselines showed excellent correlation with the experimental data, confirmed by high Interclass Correlation Coefficients of 0.995–0.998, and enabled the quantification of pyocyanin – with linearity extended down to 0.18 μM in Human Serum and 0.336 μM in both Britton-Robinson buffer and Simulated Wound Fluid, and derived Limits of Detection of 0.17, 0.15 and 0.09 μM, respectively, in this proof-of-concept study. Therefore, the use of very simple, cost-effective printed carbon materials enabled the detection of clinically relevant concentrations of this important biomarker through a new baseline fitting model and offers a novel approach for point-of-care diagnostics where Pseudomonas aeruginosa infections are critical.

Hydrogen peroxide has important roles within cellular functions, as a prevalent form of Reactive Oxygen Species, detection within mammalian cells is of metabolic importance; typically requiring cell lysis or fluorescence-based methods to quantify. Herein, we explore the novel use of Prussian blue mediated, pad printed carbon electrodes to allow the indirect detection of cellular peroxides in bulk culture media, which facilitates non-invasive, real-time detection. Electrodes demonstrated capacity to detect H2O2 with a linear range of 1-200 μM in CMEM (R

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In this communication, we argue about the need for an extensive investigation of the relationship between body fatness and fat distribution and experimental pain to explore the factors that might contribute to the increased prevalence of pain conditions in obese individuals.

The measurement of pH is important throughout many biological systems, but there are limited available technologies to enable its periodical monitoring in the complex, small volume, media often used in cell culture experiments across a range of disciplines. Herein, pad printed electrodes are developed and characterised through modification with: a commercially available fullerene multiwall carbon nanotube composite applied in Nafion, casting of hydrophobic ubiquinone as a pH probe to provide the electrochemical signal, and coated in Polyethylene glycol to reduce fouling and potentially enhance biocompatibility, which together are proven to enable the determination of pH in cell culture media containing serum. The ubiquinone oxidation peak position (Epa) provided an indirect marker of pH across the applicable range of pH 6–9 (R2 = 0.9985, n = 15) in complete DMEM. The electrochemical behaviour of these sensors was also proven to be robust; retaining their ability to measure pH in cell culture media supplemented with serum up to 20% (v/v) [encompassing the range commonly employed in cell culture], cycled > 100 times in 10% serum containing media and maintain > 60% functionality after 5 day incubation in a 10% serum containing medium. Overall, this proof of concept research highlights the potential applicability of this, or similar, electrochemical approaches to enable to detection or monitoring of pH in complex cell culture media.

© 2018 Astita et al. Background: The relationship between obesity and pain remains unclear. The aim of this systematic review was to determine whether response to experimentally-evoked pain differed between obese and non-obese individuals. Studies that compared responses to experimentally-evoked pain between obese and non-obese human participants post-puberty (i.e. >16 years) were sought. Eligible studies published between January 1950 and May 2017 were identified by searching OVID, MEDLINE, EMBASE and Science Direct. Explanation: Methodological quality of included studies was assessed using the ‘QualSyst’ questionnaire. Of 1106 references identified only nine studies (683 participants) were eligible for review. Pressure pain was assessed in five studies and electrical pain in three studies. Two studies investigated thermal pain. Obesity was categorized according to body mass index (BMI) or as weight as a percentage of ideal body weight. Six of the nine included studies were of low methodological quality. There was a lack of extractable data to pool for meta-analysis of studies using thermal or electrical pain. A forest plot of data extracted from four studies on pressure pain threshold found no differences between obese and non-obese groups (overall effect size was Z=0.57, p=0.57). Conclusion: Small sample size was the main limitation in all studies. Participants with obesity were more sensitive to mechanical noxious stimuli than non-obese participants in three of five studies. However, overall, it was not possible to determine whether there are differences in pain sensitivity response to experimental stimuli between obese and non-obese individuals.

Background and aims: Obese individuals have increased concentrations of pro-inflammatory cytokines and some other markers of inflammationand an increased risk of metabolic disorders. The relationship between obesity, pro-inflammatory cytokines and pain sensitivity response is not fully understood. Aim of the Study:To investigate associations between body fat distribution, C reactive protein (CRP), interleukin 6 (IL-6), tumour necrosis factor alpha (TNF-a), leptin and pain sensitivity in adults. Methods: 38 adults (n=18 women) were grouped as normal weight(n=22) or obese (n=16), based on body mass index (BMI).Measurements of pressure pain and cold pressor pain sensitivity response, biomarkers (venous blood), and body composition (dual X-ray absorptiometry) were evaluated for each participant. Results: Pressure pain threshold was significantly lower in obese(mean+SD=340.93±93.58 kpa)compared to the normal weight group(447.45±203.72 kpa, p=0.039, t-test). Forward regression suggested that high gynoid fat (g)was associated with lower pressure pain thresholds (ß=-0.383, p=0.028), high lower limb fat (g) was associated with lower cold pain thresholds (ß=-0.495, p=0.003)and high IL-6 predicted higher cold pain tolerance (ß=0.345, p = 0.049). Women were more sensitive to pressure pain (P=0.03). Conclusion: Gynoid and lower limb fat content correlated with pain sensitivity response in adults, whereby those with greater fat content were more sensitive to pain. However, this may be a reflection of the sex differences in pain sensitivity as women have greater gynoid and leg fat contents.

BACKGROUND: The aim of this study was to investigate the influence of body fat percentage and its distribution on sensory detection and pain sensitivity responses to experimentally induced noxious stimuli in otherwise pain-free individuals. METHODS: Seventy-two participants were divided into three equal groups according to their body mass index (BMI: normal, overweight and obese). Percentage body fat was estimated using a four-site skinfold method. Measurements of cold pressor pain threshold, tolerance and intensity; contact thermal sensory detection and heat pain threshold and tolerance (TSA-II - NeuroSensory Analyzer, Medoc); and blunt pressure pain threshold (algometer, Somedic SenseLab AB) were taken at the waist and thenar eminence. RESULTS: Mean ± SD pressure pain threshold of the obese group (620.72 ± 423.81 kPa) was significantly lower than normal (1154.70 ± 847.18 kPa) and overweight (1285.14 ± 998.89 kPa) groups. Repeated measures ANOVA found significant effects for site for cold detection threshold (F1,68  = 8.3, p = 0.005) and warm detection threshold (F1,68  = 38.69, p = 0.001) with waist having lower sensory detection thresholds than thenar eminence. For heat pain threshold, there were significant effects for site (F1,68  = 4.868, p = 0.031) which was lower for waist compared with thenar eminence (mean difference = 0.89 °C). CONCLUSION: Obese individuals were more sensitive than non-obese individuals to pressure pain but not to thermal pain. Body sites may vary in their response to different types and intensities of stimuli. The inconsistency of findings within and between research studies should catalyse further research in this field. SIGNIFICANCE: This study provided evidence that body mass index and distribution of body fat can influence sensory detection and pain sensitivity. Obese individuals were more sensitive than normal range body mass index individuals to pressure pain but not to thermal pain. Pain response varied according to subcutaneous body fat at different body sites. These findings strengthen arguments that weight loss should be a significant aspect of a pain management programme for obese pain patients.