A low-cost internal standard loader for solid-phase sorbing tools.

Solid-phase sorption is widely used for the analysis of gaseous specimens as it allows at the same time to preconcentrate target analytes and store samples for relatively long periods. The addition of internal standards (ISs) in the analytical workflow can greatly reduce the variability of the analyses and improve the reliability of the protocols. In this work, we describe the development and testing of a portable system for the reliable production of gaseous mixture of8D-Toluene in a 1L Silonite canister as well as its reproducible loading into solid-phase sorbing tools as ISs. The portable system was tested using needle trap microextraction, solid-phase extraction, and thin-film microextraction techniques commonly employed for the analysis of gaseous samples. Even though our specific interest is in breath analysis, the system can also be used for the collection of any kind of gaseous specimen. A microcontroller allows the fine control of the sampling flow by a digital mass flow controller. Flow rate and sample volume could be set either through a rotary encoder mounted onto the control board or through a dedicated android app. The variability of the airflow is in the range 5-200 ml min-1and it is lower than 1%, whereas the variability of the IS (8D-Toluene) concentration dispensed over time by the loader measured by selected-ion flow-tube mass spectrometry (MS) is <3%. This combination resulted in intra- and inter-day precision of the amount loaded in the sorbent tools lower than 15%. No carry-over was detected in the loader after the delivery of the8D-Toluene measured by gas chromatography-MS. The8D-Toluene concentration in the canister was stable for up to three weeks at room temperature.

Sweat analysis with a wearable sensing platform based on laser-induced graphene.

The scientific community has shown increasing interest in laser scribing for the direct fabrication of conductive graphene-based tracks on different substrates. This can enable novel routes for the noninvasive analysis of biofluids (such as sweat or other noninvasive matrices), whose results can provide the rapid evaluation of a person's health status. Here, we present a wearable sensing platform based on laser induced graphene (LIG) porous electrodes scribed on a flexible polyimide sheet, which samples sweat through a paper sampler. The device is fully laser manufactured and features a two layer design with LIG-based vertical interconnect accesses. A detailed characterization of the LIG electrodes including pore size, surface groups, surface area in comparison to electroactive surface area, and the reduction behavior of different LIG types was performed. The bare LIG electrodes can detect the electrochemical oxidation of both uric acid and tyrosine. Further modification of the surface of the LIG working electrode with an indoaniline derivative [4-((4-aminophenyl)imino)-2,6-dimethoxycyclohexa-2,5-dien-1-one] enables the voltammetric measurement of pH with an almost ideal sensitivity and without interference from other analytes. Finally, electrochemical impedance spectroscopy was used to measure the concentrations of ions through the analysis of the sweat impedance. The device was successfully tested in a real case scenario, worn on the skin during a sports session. In vitro tests proved the non-cytotoxic effect of the device on the A549 cell line.

The peppermint breath test: a benchmarking protocol for breath sampling and analysis using GC-MS.

Exhaled breath contains hundreds of volatile organic compounds (VOCs) which offer the potential for diagnosing and monitoring a wide range of diseases. As the breath research field has grown, sampling and analytical practices have become highly varied between groups. Standardisation would allow meta-analyses of data from multiple studies and greater confidence in published results. Washout of VOCs from ingestion into the blood and subsequently breath could provide data for an initial assessment of inter-group performance. The Peppermint Initiative has been formed to address this task of standardisation. In the current study we aimed to generate initial benchmark values for thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS) analysis of breath samples containing peppermint-derived VOCs using data from three independent European research groups. Initially, headspace analysis of peppermint oil capsules was performed to determine compounds of interest. Ten healthy participants were recruited by each three groups across Europe. The standard Peppermint protocol was followed. In brief, each participant provided a baseline breath sample prior to taking a peppermint capsule, with further samples collected at 60, 90, 165, 285 and 360 min following ingestion. Sampling and analytical protocols were different for each group, in line with their usual practice. Samples were analysed by TD-GC-MS and benchmarking values determined for the time taken for detected peppermint VOCs to return to baseline values. Sixteen compounds were identified in the capsule headspace, and all were confirmed in breath following ingestion of the peppermint capsules. Additionally, 2,3-dehydro-1,8-cineole was uniquely found in the breath samples, with a washout profile that suggested it was a product of metabolism of peppermint compounds. Five compounds (α-pinene, ß-pinene, eucalyptol, menthol and menthone) were quantified by all three groups. Differences were observed between the groups, particularly for the recovery of menthone and menthol. The average time taken for VOCs to return to baseline was selected as the benchmark and were 377, 423, 533, 418 and 336 min for α-pinene, ß-pinene, eucalyptol, menthone and menthol respectively. We have presented an initial set of easy-to-measure benchmarking values for assessing the performance of TD-GC-MS systems for the analysis of VOCs in breath. These values will be updated when more groups provide additional data.

Decision-making process as guide to the management of non-carious cervical lesions with and without painful symptomatology.

The aim of this study is to develop a decision-making path for the management of non-carious cervical lesions (NCCLs) associated with or without dentin hypersensitivity (DH) This will allow to limit or delay invasive approaches identifying the causes that produced them. The need for this review is because there are no clear guidelines in the current literature for the treatment of NCCLs. Usually, the selection of the best therapy option is postponed to clinical judgment which can be influenced by a patient's demands (aesthetic, symptomatologic reasons or worsening of pre-existing NCCL). To establish a therapeutic plan the young dentist should be able to distinguish the NCCLs that need to be monitored over time from those in need of early treatment. Indeed, the experience of the dentist and the compliance of the patient play a decisive role for the success of the therapy.

A Biosensor for the Detection of Acetylcholine and Diazinon.

Acetylcholine is a neurotransmitter and a neuromodulator found in the autonomic, peripheral and central nervous systems. Diazinon is a pesticide with toxic effects on humans, such as the inhibition of acetylcholine. In this paper, a biosensor is proposed for the detection of acetylcholine (range 70 - 1000 µM) and diazinon (range 0.3 - 20000 ppb). This biosensor combines a pH-sensitive layer of reduced graphene oxide functionalized with 4-aminobenzoic acid and acetylcholinesterase. This enzyme was immobilized on reduced graphene oxide and it catalyzed the conversion of acetylcholine into choline and acetic acid, locally decreasing the pH value and triggering the sensor response. The limit of detection for the acetylcholine and diazinon were 70 µM and 0.3 ppb, respectively.

A sampler prototype for the simultaneous collection of exhaled air and breath condensate.

Exhaled air and breath condensate contain a large number of health biomarkers, such as volatile and semi-volatile organic compounds, proteins and lipids. Nowadays, the collection of breath samples is carried out by commercial or lab-made sampling systems that collect only one type of sample (e.g. gaseous or condensate phase), thus limiting the diagnostic capability of breath tests. This work presents a portable prototype optimized for the simultaneous collection of gaseous exhaled breath and exhaled breath condensate within five minutes. The system is fully portable and has a total weight of about 1 Kg. An illustrative determination of ethanol, isoprene, acetone, isopropyl alcohol, 1-propanol, 2-butanone, 2-pentanone, toluene and xylenes in breath, and cortisol and 8-iso-prostaglandin F2α in breath condensate is discussed.

An innovative pick-up impression technique.

Precision of impression is one of the keys for the success of implant-prosthetic rehabilitation. The choice of correct techniques, performance materials, and operator skills, can improve the accuracy of impression. The proposed technique describes a modified pick-up impression technique, both for single and multiple implants, aiming to combine the advantages of direct technique (open-tray or pick-up impression) with those of indirect technique (closed-tray or transfer type impression technique) in order to improve the accuracy of implant impression.

Determination of carbonyl compounds in exhaled breath by on-sorbent derivatization coupled with thermal desorption and gas chromatography-tandem mass spectrometry.

A reliable method for the determination of carbonyl compounds in exhaled breath based on on-sorbent derivatization coupled with thermal desorption and gas chromatography-tandem mass spectrometry is described. The analytical performances were optimized for a mixture of C2-C9 aldehydes and C3-C9 ketones, particularly interesting for clinical applications, by using an internal standard and applying a 23 full factorial design. A volume of sample (250 ml) was loaded at 50 ml min-1 into a Tenax GR sorbent tube containing 130 nmol of O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride. All compounds showed a limit of detection lower than 200 pptv. The yield of the derivatization procedure was normalized by adding to the sample a known amount of 6D-acetone as an internal standard. This allowed halving the relative standard deviation to 10% and 15% for the mono-and di-carbonyl compounds, respectively, thus improving reliability. The optimized method was applied to the determination of carbonyl compounds in 12 breath samples collected from four patients suffering from heart failure during hospitalization.

Determination of volatile organic compounds in exhaled breath of heart failure patients by needle trap micro-extraction coupled with gas chromatography-tandem mass spectrometry.

The analytical performances of needle trap micro-extraction (NTME) coupled with gas chromatography-tandem mass spectrometry were evaluated by analyzing a mixture of twenty-two representative breath volatile organic compounds (VOCs) belonging to different chemical classes (i.e. hydrocarbons, ketones, aldehydes, aromatics and sulfurs). NTME is an emerging technique that guarantees detection limits in the pptv range by pre-concentrating low volumes of sample, and it is particularly suitable for breath analysis. For most VOCs, detection limits between 20 and 500 pptv were obtained by pre-concentrating 25 ml of a humidified standard gas mixture at a flow rate of 15 ml min-1. For all compounds, inter- and intra-day precisions were always below 15%, confirming the reliability of the method. The procedure was successfully applied to the analysis of exhaled breath samples collected from forty heart failure (HF) patients during their stay in the University Hospital of Pisa. The majority of patients (about 80%) showed a significant decrease of breath acetone levels (a factor of 3 or higher) at discharge compared to admission (acute phase) in correspondence to the improved clinical conditions during hospitalization, thus making this compound eligible as a biomarker of HF exacerbation.

Temperature and pH sensors based on graphenic materials.

Point-of-care applications and patients' real-time monitoring outside a clinical setting would require disposable and durable sensors to provide better therapies and quality of life for patients. This paper describes the fabrication and performances of a temperature and a pH sensor on a biocompatible and wearable board for healthcare applications. The temperature sensor was based on a reduced graphene oxide (rGO) layer that changed its electrical resistivity with the temperature. When tested in a human serum sample between 25 and 43°C, the sensor had a sensitivity of 110±10Ω/°C and an error of 0.4±0.1°C compared with the reference value set in a thermostatic bath. The pH sensor, based on a graphene oxide (GO) sensitive layer, had a sensitivity of 40±4mV/pH in the pH range between 4 and 10. Five sensor prototypes were tested in a human serum sample over one week and the maximum deviation of the average response from reference values obtained by a glass electrode was 0.2pH units. For biological applications, the temperature and pH sensors were successfully tested for in vitro cytotoxicity with human fibroblast cells (MRC-5) over 24h.

A D-optimal design to model the performances of dressings and devices for negative pressure wound therapy.

A D-optimal design was used to identify and model variables that affect the transit time of wound exudate through an illustrative dressing used for negative pressure wound therapy. Many authors have addressed the clinical benefits of negative pressure wound therapy, but limited information is available on how to assess performances of dressings. In this paper, the transit time of wound exudate through a dressing was chosen as a model parameter to show how experimental design (DOE) can be used for this purpose. Results demonstrated that rate of exudate production, temperature and dressing thickness were the variables with the largest impact on transit time. The DOE approach could be used to model other dressing properties, like for example capability of absorbing excess exudate or breathability.

A graphene oxide pH sensor for wound monitoring.

This article describes the fabrication and characterization of a pH sensor for monitoring the wound status. The pH sensitive layer consists of a graphene oxide (GO) layer obtained by drop-casting 5 µÎ of GO dispersion onto the working electrode of a screen-printed substrate. Sensitivity was 31.8 mV/pH with an accuracy of 0.3 unit of pH. Open-circuit potentiometry was carried out to measure pH in an exudate sample. The GO pH sensor proved to be reliable as the comparison with results obtained from a standard glass electrode pH-meter showed negligible differences (<; 0.09 pH units in the worst case) for measurements performed over a period of 4 days.

Comparison of sampling bags for the analysis of volatile organic compounds in breath.

Nalophan, Tedlar and Cali-5-Bond polymeric bags were compared to determine the most suitable type for breath sampling and storage when volatile organic compounds are to be determined. Analyses were performed by thermal desorption gas chromatography mass spectrometry. For each bag, the release of contaminants and the chemical stability of a gaseous standard mixture containing eighteen organic compounds, as well as the CO2 partial pressure were assessed. The selected compounds were representative of breath constituents and belonged to different chemical classes (i.e. hydrocarbons, ketones, aldehydes, aromatics, sulfurs and esters). In the case of Nalophan, the influence of the surface-to-volume ratio, related to the bag's filling degree, on the chemical stability was also evaluated. Nalophan bags were found to be the most suitable in terms of contaminants released during storage (only 2-methyl-1,3-dioxalane), good sample stability (up to 24 h for both dry and humid samples), and very limited costs (about 1 € for a 20 liter bag). The (film) surface-to-(sample) volume ratio was found to be an important factor affecting the stability of selected compounds, and therefore we recommended to fill the bag completely.

Splitting livers: Trans-hilar or trans-umbilical division? Technical aspects and comparative outcomes.

UNLABELLED: Controversy remains about the best line of division for liver splitting, through Segment IV or through the umbilical fissure. Both techniques are currently used, with the choice varying between surgical teams in the absence of an evidence-based choice. We conducted a single-center retrospective analysis of 47 left split liver grafts that were procured with two different division techniques: "classical" (N = 28, Group A) or through the umbilical fissure and plate (N = 19, Group B). The allocation of recipients to each group was at random; a single transplant team performed all transplantations. Demographics, characteristics, technical aspects, and outcomes were similar in both groups. The grafts in Group A, prepared with the classical technique, were procured more often with a single BD orifice compared with the grafts in Group B; however, this was not associated with a higher incidence of biliary problems in this series of transplants (96% actual graft survival rate [median ± s.d. FOLLOW-UP: 26 ± 20 months]). Both techniques provide good quality split grafts and an excellent outcome; surgical expertise with a given technique is more relevant than the technique itself. The classical technique, however, seems to be more flexible in various ways, and surgeons may find it to be preferable.

Roux-en-Y hepatico-jejunostomy for a left segmental graft: Do not twist the loop, stick it!

Biliary complications remain a major challenge for long-term success after LT, as it is, as a rule, the most common technical - early and late - complication that occurs, and because these complications contribute to a significant number of late graft losses and retransplantations. In the pediatric age group, both biliary atresia, as the patient's condition, and the use of a left liver graft, obtained by a liver division technique, make it necessary for the use of a Roux-en-Y jejunal loop for the biliary reconstruction in the majority of cases. A slight modification of the technique is presented, consisting of a straight positioning along the cut surface (rather than the conventional position that results in a harpoon shape). A favorable outcome in terms of a technical complication and graft survival was observed. This way of doing this is an interesting variation and adds to the surgical armamentarium.

A dual mode breath sampler for the collection of the end-tidal and dead space fractions.

This work presents a breath sampler prototype automatically collecting end-tidal (single and multiple breaths) or dead space air fractions (multiple breaths). This result is achieved by real time measurements of the CO2 partial pressure and airflow during the expiratory and inspiratory phases. Suitable algorithms, used to control a solenoid valve, guarantee that a Nalophan(®) bag is filled with the selected breath fraction even if the subject under test hyperventilates. The breath sampler has low pressure drop (<0.5 kPa) and uses inert or disposable components to avoid bacteriological risk for the patients and contamination of the breath samples. A fully customisable software interface allows a real time control of the hardware and software status. The performances of the breath sampler were evaluated by comparing (a) the CO2 partial pressure calculated during the sampling with the CO2 pressure measured off-line within the Nalophan(®) bag; (b) the concentrations of four selected volatile organic compounds in dead space, end-tidal and mixed breath fractions. Results showed negligible deviations between calculated and off-line CO2 pressure values and the distributions of the selected compounds into dead space, end-tidal and mixed breath fractions were in agreement with their chemical-physical properties.

A breath sampling system assessing the influence of respiratory rate on exhaled breath composition.

This work presents a computerized system to monitor mouth pressure, tidal volume, exhaled airflow, respiration rate and end-tidal partial pressure of CO2 during breath collection. The system was used to investigate the effect of different respiratory rates on the volatile organic compounds (VOCs) concentrations in exhaled breath. For this purpose, VOCs with well-defined biochemical pathways and different chemical and physical properties were selected as biomarkers related to metabolism (acetone and isopropyl alcohol), cholesterol synthesis (isoprene) and intestinal microflora activity (ethanol). Mixed breath was collected from a nominally healthy volunteer in resting conditions by filling a Nalophan bag. The subject followed a regimented breathing pattern at different respiratory rates (10, 30 and 50 breaths per minute). Results highlight that ventilation pattern strongly influences the concentration of the selected compounds. The proposed system allows exhaled breath to be collected also in patients showing dyspnea such as in case of chronic heart failure, asthma and pulmonary diseases.

Post-operative elimination of sevoflurane anesthetic and hexafluoroisopropanol metabolite in exhaled breath: pharmacokinetic models for assessing liver function.

Sevoflurane (SEV), a commonly used anesthetic agent for invasive surgery, is directly eliminated via exhaled breath and indirectly by metabolic conversion to inorganic fluoride and hexafluoroisopropanol (HFIP), which is also eliminated in the breath. We studied the post-operative elimination of SEV and HFIP of six patients that had undergone a variety of surgeries lasting between 2.5 to 8.5 h using exhaled breath analysis. A classical three compartments pharmacokinetic model developed for the study of environmental contaminants was fitted to the breath data. We found that SEV kinetic behavior following surgery (for up to six days) is consistent across all subjects whereas the production and elimination of HFIP varies to some extent. We developed subject specific parameters for HFIP metabolism and interpreted the differences in the context of timing and dose of anesthesia, type of surgery, and specific host factors. We propose methods for assessing individual patient liver function using SEV as a probe molecule for assessing efficiency of liver metabolism to HFIP. This work is valuable not only for the clinical study of metabolism recovery, but potentially also for the study of the interaction of other manufactured and environmental compounds with human systems biology in controlled exposure and observational studies.

Monitoring breath during oral glucose tolerance tests.

The evolution of breath composition during oral glucose tolerance tests (OGTTs) was analysed by thermal desorption/gas chromatography/mass spectrometry in 16 subjects and correlated to blood glucose levels. The glucose tolerance tests classified five of the subjects as diabetics, eight as affected by impaired glucose tolerance and three as normoglycaemic. Acetone levels were generally higher in diabetics (average concentration values: diabetics, 300 ± 40 ppbv; impaired glucose tolerance, 350 ± 30 ppbv; normoglycaemic, 230 ± 20 ppbv) but the large inter-individual variability did not allow us to identify the three groups by this parameter alone. The exhalation of 3-hydroxy-butan-2-one and butane-2,3-dione, likely due to the metabolization of glucose by bacteria in the mouth, was also observed. Future work will involve the extension of the analyses to other volatile compounds by attempting to improve the level of discrimination between the various classes of subjects.