Urinary Volatile Organic Compound Testing in Fast-Track Patients with Suspected Colorectal Cancer.

Colorectal symptoms are common but only infrequently represent serious pathology, including colorectal cancer (CRC). A large number of invasive tests are presently performed for reassurance. We investigated the feasibility of urinary volatile organic compound (VOC) testing as a potential triage tool in patients fast-tracked for assessment for possible CRC. A prospective, multi-center, observational feasibility study was performed across three sites. Patients referred to NHS fast-track pathways for potential CRC provided a urine sample that underwent Gas Chromatography-Mass Spectrometry (GC-MS), Field Asymmetric Ion Mobility Spectrometry (FAIMS), and Selected Ion Flow Tube Mass Spectrometry (SIFT-MS) analysis. Patients underwent colonoscopy and/or CT colonography and were grouped as either CRC, adenomatous polyp(s), or controls to explore the diagnostic accuracy of VOC output data supported by an artificial neural network (ANN) model. 558 patients participated with 23 (4%) CRC diagnosed. 59% of colonoscopies and 86% of CT colonographies showed no abnormalities. Urinary VOC testing was feasible, acceptable to patients, and applicable within the clinical fast track pathway. GC-MS showed the highest clinical utility for CRC and polyp detection vs. controls (sensitivity = 0.878, specificity = 0.882, AUROC = 0.896) but it is labour intensive. Urinary VOC testing and analysis are feasible within NHS fast-track CRC pathways. Clinically meaningful differences between patients with cancer, polyps, or no pathology were identified suggesting VOC analysis may have future utility as a triage tool.

Investigation of urinary volatile organic compounds as novel diagnostic and surveillance biomarkers of bladder cancer.

BACKGROUND: The diagnosis and surveillance of urothelial bladder cancer (UBC) require cystoscopy. There is a need for biomarkers to reduce the frequency of cystoscopy in surveillance; urinary volatile organic compound (VOC) analysis could fulfil this role. This cross-sectional study compared the VOC profiles of patients with and without UBC, to investigate metabolomic signatures as biomarkers. METHODS: Urine samples were collected from haematuria clinic patients undergoing diagnostic cystoscopy and UBC patients undergoing surveillance. Urinary headspace sampling utilised solid-phase microextraction and VOC analysis applied gas chromatography-mass spectrometry; the output underwent metabolomic analysis. RESULTS: The median participant age was 70 years, 66.2% were male. Of the haematuria patients, 21 had a new UBC diagnosis, 125 had no cancer. In the surveillance group, 75 had recurrent UBC, 84 were recurrence-free. A distinctive VOC profile was observed in UBC patients compared with controls. Ten VOCs had statistically significant abundances useful to classify patients (false discovery rate range 1.9 × 10-7-2.8 × 10-2). Two prediction models were evaluated using internal validation. An eight-VOC diagnostic biomarker panel achieved AUROC 0.77 (sensitivity 0.71, specificity 0.72). A six-VOC surveillance biomarker panel obtained AUROC 0.80 (sensitivity 0.71 and specificity 0.80). CONCLUSIONS: Urinary VOC analysis could aid the diagnosis and surveillance of UBC.

Application of a solid-phase microextraction-gas chromatography-mass spectrometry/metal oxide sensor system for detection of antibiotic susceptibility in urinary tract infection-causing Escherichia coli - A proof of principle study.

PURPOSE: Antibiotic resistance is widespread throughout the world and represents a serious health concern. There is an urgent need for the development of novel tools for rapidly distinguishing antibiotic resistant bacteria from susceptible strains. Previous work has demonstrated that differences in antimicrobial susceptibility can be reflected in differences in the profile of volatile organic compounds (VOCs) produced by dissimilar strains. The aim of this study was to investigate the effect of the presence of cephalosporin antibiotics on the VOC profile of extended spectrum beta-lactamase (ESBL) and non-ESBL producing strains of Escherichia coli. MATERIAL AND METHODS: In this study, VOCs from strains of Escherichia coli positive and negative for the most commonly encountered ESBL, CTX-M in the presence of cephalosporin antibiotics were assessed using solid-phase microextraction (SPME) coupled with a combined gas chromatography-mass spectrometry/metal oxide sensor (GC-MS/MOS) system. RESULTS: Our proof-of-concept study allowed for distinguishing CTX-M positive and negative bacteria within 2 â€‹h after the addition of antibiotics. One MOS signal (RT: 22.6) showed a statistically significant three-way interaction (p â€‹= â€‹0.033) in addition to significant two-way interactions for culture and additive (p â€‹= â€‹0.046) plus time and additive (p â€‹= â€‹0.020). There were also significant effects observed for time (p â€‹= â€‹0.009), culture (p â€‹= â€‹0.030) and additive (p â€‹= â€‹0.028). No effects were observed in the MS data. CONCLUSIONS: The results of our study showed the potential of VOC analysis using SPME combined with a GC-MS/MOS system for the early detection of CTX-M-producing, antibiotic-resistant E. coli, responsible for urinary tract infections (UTIs).

Hyphenated Mass Spectrometry versus Real-Time Mass Spectrometry Techniques for the Detection of Volatile Compounds from the Human Body.

Mass spectrometry (MS) is an analytical technique that can be used for various applications in a number of scientific areas including environmental, security, forensic science, space exploration, agri-food, and numerous others. MS is also continuing to offer new insights into the proteomic and metabolomic fields. MS techniques are frequently used for the analysis of volatile compounds (VCs). The detection of VCs from human samples has the potential to aid in the diagnosis of diseases, in monitoring drug metabolites, and in providing insight into metabolic processes. The broad usage of MS has resulted in numerous variations of the technique being developed over the years, which can be divided into hyphenated and real-time MS techniques. Hyphenated chromatographic techniques coupled with MS offer unparalleled qualitative analysis and high accuracy and sensitivity, even when analysing complex matrices (breath, urine, stool, etc.). However, these benefits are traded for a significantly longer analysis time and a greater need for sample preparation and method development. On the other hand, real-time MS techniques offer highly sensitive quantitative data. Additionally, real-time techniques can provide results in a matter of minutes or even seconds, without altering the sample in any way. However, real-time MS can only offer tentative qualitative data and suffers from molecular weight overlap in complex matrices. This review compares hyphenated and real-time MS methods and provides examples of applications for each technique for the detection of VCs from humans.

An Optimization of Liquid-Liquid Extraction of Urinary Volatile and Semi-Volatile Compounds and Its Application for Gas Chromatography-Mass Spectrometry and Proton Nuclear Magnetic Resonance Spectroscopy.

Urinary volatile compounds (VCs) have been recently assessed for disease diagnoses. They belong to very diverse chemical classes, and they are characterized by different volatilities, polarities and concentrations, complicating their analysis via a single analytical procedure. There remains a need for better, lower-cost methods for VC biomarker discovery. Thus, there is a strong need for alternative methods, enabling the detection of a broader range of VCs. Therefore, the main aim of this study was to optimize a simple and reliable liquid-liquid extraction (LLE) procedure for the analysis of VCs in urine using gas chromatography-mass spectrometry (GC-MS), in order to obtain the maximum number of responses. Extraction parameters such as pH, type of solvent and ionic strength were optimized. Moreover, the same extracts were analyzed using Proton Nuclear Magnetic Resonance Spectroscopy (1H-NMR), to evaluate the applicability of a single urine extraction for multiplatform purposes. After the evaluation of experimental conditions, an LLE protocol using 2 mL of urine in the presence of 2 mL of 1 M sulfuric acid and sodium sulphate extracted with dichloromethane was found to be optimal. The optimized method was validated with the external standards and was found to be precise and linear, and allowed for detection of >400 peaks in a single run present in at least 50% of six samples-considerably more than the number of peaks detected by solid-phase microextracton fiber pre-concentration-GC-MS (328 ± 6 vs. 234 ± 4). 1H-NMR spectroscopy of the polar and non-polar extracts extended the range to >40 more (mainly low volatility compounds) metabolites (non-destructively), the majority of which were different from GC-MS. The more peaks detectable, the greater the opportunity of assessing a fingerprint of several compounds to aid biomarker discovery. In summary, we have successfully demonstrated the potential of LLE as a cheap and simple alternative for the analysis of VCs in urine, and for the first time the applicability of a single urine solvent extraction procedure for detecting a wide range of analytes using both GC-MS and 1H-NMR analysis to enhance putative biomarker detection. The proposed method will simplify the transport between laboratories and storage of samples, as compared to intact urine samples.

Breath analysis for detection of viral infection, the current position of the field.

The COVID-19 pandemic has highlighted the importance of rapid, cost effective, accurate, and non-invasive testing for viral infections. Volatile compounds (VCs) have been suggested for several decades as fulfilling these criteria. However currently very little work has been done in trying to diagnose viral infections using VCs. Much of the work carried out to date involves the differentiation of bacterial and viral sources of infection and often the detection of bacterial and viral co-infection. However, this has usually been done in vitro and very little work has involved the use of human participants. Viruses hijack the host cell metabolism and do not produce their own metabolites so identifying virus specific VCs is at best a challenging task. However, there are proteins and lipids that are potential candidates as markers of viral infection. The current understanding is that host cell glycolysis is upregulated under viral infection to increase the available energy for viral replication. There is some evidence that viral infection leads to the increase of production of fatty acids, alkanes, and alkanes related products. For instance, 2,3-butandione, aldehydes, 2,8-dimethyl-undecane and n-propyl acetate have all been correlated with viral infection. Currently, the literature points to markers of oxidative stress (e.g. nitric oxide, aldehydes etc) being the most useful in the determination of viral infection. The issue, however, is that there are also many other conditions that can lead to oxidative stress markers being produced. In this review a range of (mainly mass spectrometric) methods are discussed for viral detection in breath, including breath condensate. Currently MALDI-ToF-MS is likely to be the preferred method for the identification of viral strains and variants of those strains, however it is limited by its need for the viral strains to have been sequenced and logged in a database.

A mechanistic study and review of volatile products from peroxidation of unsaturated fatty acids: an aid to understanding the origins of volatile organic compounds from the human body.

The assessment of volatile compounds (VOCs) for disease diagnosis is a growing area of research. There is a need to provide hard evidence i.e. biochemical routes, to justify putative VOC biomarkers, as in many cases this remains uncertain, which weakens their authenticity. Recently reports of volatile hydrocarbons and or aldehydes in bodily fluids and breath have been attributed to oxidative stress, although as discussed here, fewer compounds have been reported than expected from a mechanistic examination. Oxidative stress can result from many disease states which produce inflammation, and a better understanding of the interconnection between oxidative stress and the release of VOCs from target diseased and healthy organs could greatly help diagnoses. It is generally considered that oxidation of unsaturated fatty acids are a major source of these VOCs. An investigation listing the many possible volatile oxidation products has not been undertaken. This is described here using a mechanistic analysis (based on the literature) of the compounds derived from molecular cleavage and the results compared with a recent review of all the VOCs emanating from the human body, which satisfactorily explains the presence of at least 100 VOCs. Six important unsaturated fatty acids, oleic, palmitoleic, linoleic, linolenic, arachidonic, and cervonic acids have been shown to be capable of producing up to 18 n+6 unique breakdown products (where n = the number of alkene double bonds in the fatty acid hydrocarbon chain), in total 299 compounds. In many cases these have not been reported. We suggest several reasons for this: these VOCs have not been expected, so researchers are not looking for them and importantly some are not present in the mass spectral libraries, or they are too low a concentration to have been detected, or are not present. Furthermore a theoretical explanation for the origins of branched aldehydes and other compounds arising from bacterial oxidative metabolism of unsaturated fatty acids are described.

Contactless sensing of liquid marbles for detection, characterisation & computing.

Liquid marbles (LMs) are of growing interest in many fields, including microfluidics, microreactors, sensors, and signal carriers. The generation of LMs is generally performed manually, although there has recently been a burst of publications involving 'automatic marble makers'. The characteristics of a LM is dependent on many things, including how it is generated, it is therefore important to be able to characterise LMs once made. Here is presented a novel contactless LM sensor, constructed on a PCB board with a comb-like structure of 36 interlacing electrical traces, 100 µm wide and 100 µm apart. This cheap, scalable, and easy to use sensor exploits the inherent impedance (comprised of the electrical resistance, capacitive reactance and inductive reactance) of different LMs. With it, parameters of a LM can be easily determined, without interfering with the LM. These parameters are (1) particle size of the LM coating, (2) the concentration of a NaCl solution used as the LM core, and (3) the volume of the LM. Additionally, due to the comb-like nature of the sensor, the accurate positioning (down to the inter-trace spacing) of the LM can be ascertained. The new sensor has been shown to work under both static and dynamic (mobile) conditions. The capacitance of a LM was recorded to be 0.10 pF, which compares well with the calculated value of 0.12 pF.

Neuromorphic Liquid Marbles with Aqueous Carbon Nanotube Cores.

Neuromorphic computing devices attempt to emulate features of biological nervous systems through mimicking the properties of synapses toward implementing the emergent properties of their counterparts, such as learning. Inspired by recent advances in the utilization of liquid marbles (LMs, microliter quantities of fluid coated in hydrophobic powder) for the creation of unconventional computing devices, we describe the development of LMs with neuromorphic properties through the use of copper coatings and 1.0 mg mL-1 carbon nanotube (CNT)-containing fluid cores. Experimentation was performed through sandwiching the LMs between two cup-style electrodes and stimulating them with repeated dc pulses at 3.0 V. Our results demonstrate that "entrainment" of CNT-filled copper LMs via periodic pulses can cause their electrical resistance to rapidly switch between high to low resistance profiles upon inverting the polarity of stimulation: the reduction in resistance between high and low profiles was approximately 88% after two rounds of entrainment. This effect was found to be reversible through reversion to the original stimulus polarity and was strengthened by repeated experimentation, as evidenced by a mean reduction in time to switching onset of 43%. These effects were not replicated in nanotube solutions not bound inside LMs. Our electrical characterization also reveals that nanotube-filled LMs exhibit pinched loop hysteresis IV profiles consistent with the description of memristors. We conclude by discussing the applications of this technology to the development of unconventional computing devices and the study of emergent characteristics in biological neural tissue.

Thermal switch of oscillation frequency in Belousov-Zhabotinsky liquid marbles.

External control of oscillation dynamics in the Belousov-Zhabotinsky (BZ) reaction is important for many applications including encoding computing schemes. When considering the BZ reaction, there are limited studies dealing with thermal cycling, particularly cooling, for external control. Recently, liquid marbles (LMs) have been demonstrated as a means of confining the BZ reaction in a system containing a solid-liquid interface. BZ LMs were prepared by rolling 50 µl droplets in polyethylene (PE) powder. Oscillations of electrical potential differences within the marble were recorded by inserting a pair of electrodes through the LM powder coating into the BZ solution core. Electrical potential differences of up to 100 mV were observed with an average period of oscillation ca 44 s. BZ LMs were subsequently frozen to -1°C to observe changes in the frequency of electrical potential oscillations. The frequency of oscillations reduced upon freezing to 11 mHz cf. 23 mHz at ambient temperature. The oscillation frequency of the frozen BZ LM returned to 23 mHz upon warming to ambient temperature. Several cycles of frequency fluctuations were able to be achieved.

Mapping outcomes of liquid marble collisions.

Liquid marbles (LMs) have many promising roles in the ongoing development of microfluidics, microreactors, bioreactors, and unconventional computing. In many of these applications, the coalescence of two LMs is either required or actively discouraged, therefore it is important to study liquid marble collisions and establish parameters which enable the desired collision outcome. Recent reports on LM coalescence have focused on either two mobile LMs colliding, or an accelerating LM hitting a sessile LM with a backstop. A further possible scenario is the impact of a mobile LM against a non-supported static LM. This paper investigates such a collision, using high-speed videography for single-frame analysis. Multiple collisions were undertaken whilst varying the modified Weber number (We*) and offset ratios (X*). Parameter ranges of 1.0 < We* < 1.4 and 0.0 < X* < 0.1, resulted in a coalescence rate of approximately 50%. Whereas, parameter ranges X* > 0.25, and We* < 0.95 or We* > 1.55 resulted in 100% non-coalescence. Additionally, observations of LMs moving above a threshold velocity of 0.6 m s-1 have revealed a new and unusual deformation. Comparisons of the outcome of collisions whilst varying both the LM volume and the powder grain size have also been made, revealing a strong link. The results of this work provide a deeper understanding of LM coalescence, allowing improved control when designing future collision experiments.

Sniffing out resistance - Rapid identification of urinary tract infection-causing bacteria and their antibiotic susceptibility using volatile metabolite profiles.

Antibiotic resistance is set to be an unprecedented threat to modern medicine. 'Sniffing' bacteria potentially offers a rapid way to determine susceptibility. A successful proof-of-principle study is described, using thermal desorption-gas chromatography-mass spectrometry (TDGCMS) to 'smell' cephalexin and ciprofloxacin resistant and sensitive Urinary Tract Infection (UTI)-causing bacteria. 578 peaks at unique retention times were detected from 86 chromatograms of 18 bacterial isolates (E. coli, K. pneumoniae and P. aeruginosa). The isolates were grown with and without the presence of antibiotic. Chi-square analysis found 9 compounds that differed significantly between cephalexin sensitive and resistant isolates, and 22 compounds that differed significantly between ciprofloxacin sensitive and resistant isolates, at p ≤ 0.05. When antibiotic was added to the media, more differences were found in the cephalexin group, attributed to lysis, but not in the ciprofloxacin group. Further work with large sample sizes will potentially enable the development of diagnostic algorithms using presence/absence of particular compounds of interest.

Gas Chromatography Mass Spectrometry (GC-MS) Quantification of Metabolites in Stool Using 13C Labelled Compounds.

It has become increasingly important to qualitatively and quantitatively assess the volatile metabolites in a range of bodily fluids for use in monitoring health. There has been relatively little work on the quantitative analysis of compounds, particularly with respect to the effects of ethnicity or geographic location. A novel method for the quantification of compounds in stool using 13C labelled compounds as internal standards is presented. Using thermal desorption gas chromatography mass spectrometry, stool samples from 38 healthy volunteers were analysed. The 13C labelled compounds, acetone, ethyl butanoate, ethanoic acid, butanoic acid, 3-methylbutanoic acid, and indole, were added as internal standards. This process mimics the solubility characteristics of the compounds and thus the method was able to quantify the compounds within the solid stool. In total, 15 compounds were quantified: Dimethyl sulphide (26⁻25,626 ng/g), acetone (442⁻3006 ng/g), ethyl butanoate (39⁻2468 ng/g), ethyl 2-methylbutanoate (0.3⁻180 ng/g), dimethyl disulphide (35⁻1303 ng/g), 1-octen-3-one (12 ng/g), dimethyl trisulphide (10⁻410 ng/g), 1-octen-3-ol (0.4⁻58 ng/g), ethanoic acid (672⁻12,963 ng/g), butanoic acid (2493⁻11,553 ng/g), 3-methylbutanoic acid (64⁻8262 ng/g), pentanoic acid (88⁻21,886 ng/g), indole (290⁻5477 ng/g), and 3-methyl indole (37⁻3483 ng/g). Moreover, by altering the pH of the stool to pH 13 in conjunction with the addition of 13C trimethylamine, the method was successful in detecting and quantifying trimethylamine for the first time in stool samples (range 40⁻5312 ng/g). Statistical analysis revealed that samples from U.K. origin had five significantly different compounds (ethyl butanoate, 1-octen-3-ol, ethanoic acid, butanoic acid, pentanoic acid, and indole) from those of South American origin. However, there were no significant differences between vegetarian and omnivore samples. These findings are supported by pre-existing literature evidence. Moreover, we have tentatively identified 12 compounds previously not reported as having been found in stool.

Liquid Marble Actuator for Microfluidic Logic Systems.

A mechanical flip-flop actuator has been developed that allows for the facile re-routing and distribution of liquid marbles (LMs) in digital microfluidic devices. Shaped loosely like a triangle, the actuating switch pivots from one bistable position to another, being actuated by the very low mass and momentum of a LM rolling under gravity (~4 × 10-6 kg ms-1). The actuator was laser-cut from cast acrylic, held on a PTFE coated pivot, and used a PTFE washer. Due to the rocking motion of the switch, sequential LMs are distributed along different channels, allowing for sequential LMs to traverse parallel paths. This distributing effect can be easily cascaded, for example to evenly divide sequential LMs down four different paths. This lightweight, cheap and versatile actuator has been demonstrated in the design and construction of a LM-operated mechanical multiplication device - establishing its effectiveness. The actuator can be operated solely by gravity, giving it potential use in point-of-care devices in low resource areas.

Evaporation, Lifetime, and Robustness Studies of Liquid Marbles for Collision-Based Computing.

Liquid marbles (LMs) have recently attracted interest for use as cargo carriers in digital microfluidics and have successfully been implemented as signal carriers in collision-based unconventional computing circuits. Both application domains require LMs to roll over substantial distances and to survive a certain number of collisions without degrading. To evaluate the lifetime of LMs being subjected to movement and impact stresses, we have selected four types of coating to investigate: polytetrafluoroethylene (PTFE), ultrahigh density polyethylene (PE), Ni, and a mixture of Ni with PE (Ni-PE). Hierarchies of robustness have been constructed which showed that pure PE LMs survived the longest when stationary and in motion. Pure PTFE LMs were shown to be the least resilient to multiple impacts. The PTFE coating provided minimal protection against evaporative losses for small LM volumes (2 and 5 µL) however, larger LMs (10 µL) were shown to have good evaporative stabilities when stationary. Conversely, PE LMs showed a remarkable ability to withstand multiple impacts and were also stable when considering just passive evaporation. Hybrid Ni-PE LMs exhibited more resilience to multiple impacts compared to Ni LMs. Thus, when designing LM devices, it is paramount to determine impact pathways and select appropriate coating materials.

Assessment of a combined gas chromatography mass spectrometer sensor system for detecting biologically relevant volatile compounds.

There have been a number of studies in which metal oxide sensors (MOS) have replaced conventional analytical detectors in gas chromatography systems. However, despite the use of these instruments in a range of applications including breath research the sensor responses (i.e. resistance changes w.r.t. concentration of VCs) remain largely unreported. This paper addresses that issue by comparing the response of a metal oxide sensor directly with a mass spectrometer (MS), whereby both detectors are interfaced to the same GC column using an s-swafer. It was demonstrated that the sensitivity of an in-house fabricated ZnO/SnO2 thick film MOS was superior to a modern MS for the detection of a wide range of volatile compounds (VCs) of different functionalities and masses. Better techniques for detection and quantification of these VCs is valuable, as many of these compounds are commonly reported throughout the scientific literature. This is also the first published report of a combined GC-MS sensor system. These two different detector technologies when combined, should enhance discriminatory abilities to aid disease diagnoses using volatiles from e.g. breath, and bodily fluids. Twenty-nine chemical standards have been tested using solid phase micro-extraction; 25 of these compounds are found on human breath. In all but two instances the sensor exhibited the same or superior limit of detection compared to the MS. Twelve stool samples from healthy participants were analysed; the sensor detected, on average 1.6 peaks more per sample than the MS. Similarly, analysing the headspace of E. coli broth cultures the sensor detected 6.9 more peaks per sample versus the MS. This greater sensitivity is primarily a function of the superior limits of detection of the metal oxide sensor. This shows that systems based on the combination of chromatography systems with solid state sensors shows promise for a range of applications.

The use of a gas chromatography-sensor system combined with advanced statistical methods, towards the diagnosis of urological malignancies.

Prostate cancer is one of the most common cancers. Serum prostate-specific antigen (PSA) is used to aid the selection of men undergoing biopsies. Its use remains controversial. We propose a GC-sensor algorithm system for classifying urine samples from patients with urological symptoms. This pilot study includes 155 men presenting to urology clinics, 58 were diagnosed with prostate cancer, 24 with bladder cancer and 73 with haematuria and or poor stream, without cancer. Principal component analysis (PCA) was applied to assess the discrimination achieved, while linear discriminant analysis (LDA) and support vector machine (SVM) were used as statistical models for sample classification. Leave-one-out cross-validation (LOOCV), repeated 10-fold cross-validation (10FoldCV), repeated double cross-validation (DoubleCV) and Monte Carlo permutations were applied to assess performance. Significant separation was found between prostate cancer and control samples, bladder cancer and controls and between bladder and prostate cancer samples. For prostate cancer diagnosis, the GC/SVM system classified samples with 95% sensitivity and 96% specificity after LOOCV. For bladder cancer diagnosis, the SVM reported 96% sensitivity and 100% specificity after LOOCV, while the DoubleCV reported 87% sensitivity and 99% specificity, with SVM showing 78% and 98% sensitivity between prostate and bladder cancer samples. Evaluation of the results of the Monte Carlo permutation of class labels obtained chance-like accuracy values around 50% suggesting the observed results for bladder cancer and prostate cancer detection are not due to over fitting. The results of the pilot study presented here indicate that the GC system is able to successfully identify patterns that allow classification of urine samples from patients with urological cancers. An accurate diagnosis based on urine samples would reduce the number of negative prostate biopsies performed, and the frequency of surveillance cystoscopy for bladder cancer patients. Larger cohort studies are planned to investigate the potential of this system. Future work may lead to non-invasive breath analyses for diagnosing urological conditions.

Urinary Volatile Organic Compounds for the Detection of Prostate Cancer.

The aim of this work was to investigate volatile organic compounds (VOCs) emanating from urine samples to determine whether they can be used to classify samples into those from prostate cancer and non-cancer groups. Participants were men referred for a trans-rectal ultrasound-guided prostate biopsy because of an elevated prostate specific antigen (PSA) level or abnormal findings on digital rectal examination. Urine samples were collected from patients with prostate cancer (n = 59) and cancer-free controls (n = 43), on the day of their biopsy, prior to their procedure. VOCs from the headspace of basified urine samples were extracted using solid-phase micro-extraction and analysed by gas chromatography/mass spectrometry. Classifiers were developed using Random Forest (RF) and Linear Discriminant Analysis (LDA) classification techniques. PSA alone had an accuracy of 62-64% in these samples. A model based on 4 VOCs, 2,6-dimethyl-7-octen-2-ol, pentanal, 3-octanone, and 2-octanone, was marginally more accurate 63-65%. When combined, PSA level and these four VOCs had mean accuracies of 74% and 65%, using RF and LDA, respectively. With repeated double cross-validation, the mean accuracies fell to 71% and 65%, using RF and LDA, respectively. Results from VOC profiling of urine headspace are encouraging and suggest that there are other metabolomic avenues worth exploring which could help improve the stratification of men at risk of prostate cancer. This study also adds to our knowledge on the profile of compounds found in basified urine, from controls and cancer patients, which is useful information for future studies comparing the urine from patients with other disease states.

Transfer function of protoplasmic tubes of Physarum polycephalum.

The slime mould Physarum polycephalum is a large single celled myxomycete; its plasmodium consists of tubes which extend to find sources of food. It has been previously shown that the tubes are conductive with a resistance of approximately 3 MΩ, and have been used in basic DC circuits. Hybrid slime mould-electronic circuits have been proposed, using the protoplasmic tubes, grown between agar, as Physarum wires. This paper aims to evaluate the electrical properties of the protoplasmic tubes with respect to analogue and digital waveforms. The Physarum wires act as low pass filters with a mean cut off frequency of 19kHz (SD 9 KHz); they have a 12.1 dB/decade roll-off (SD 1.9 dB/decade). Mean attenuation across the band-pass range is -6 dB (S.D. 4.5 dB). The mechanism for the frequency dependant attenuation is unknown however a combination of protoplasmic electrolyte and the cytoskeletal structure is the most likely cause. The tubes last approximately 2 weeks before forming a dry sclerotia, when they cease being conductive and is the prevalent limiting factor of their practical use; this is caused by dehydration and lack of nutrition, a limitation which may be overcome. The potential for Physarum wires in hybrid circuits is strengthened; while previous circuits were simple DC circuits, this work demonstrates that they may be used as electronic components or wires in both digital and analogue circuits or even as a computing component in analogue computers.

Routing of Physarum polycephalum "signals" using simple chemicals.

In previous work the chemotaxis toward simple organic chemicals was assessed. We utilize the knowledge gained from these chemotactic assays to route Physarum polycephalum "signals" at a series of junctions. By applying chemical inputs at a simple T-junction we were able to reproducibly control the path taken by the plasmodium of P. Polycephalum. Where the chemoattractant farnesene was used at one input a routed signal could be reproducibly generated i.e., P. Polycephalum moves toward the source of chemoattractant. Where the chemoattractant was applied at both inputs the signal was reproducibly split i.e., at the junction the plasmodium splits and moves toward both sources of chemoattractant. If a chemorepellent was used then the signal was reproducibly suppressed i.e., P. Polycephalum did not reach either output and was confined to the input channel. This was regardless of whether a chemoattractant was used in combination with the chemorepellent showing a hierarchy of inhibition over attraction. If no chemical input was used in the simple circuit then a random signal was generated, whereby P. Polycephalum would move toward one output at the junction, but the direction was randomly selected.