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Article
Ligand-capped Ultrapure Metal Nanoparticle Sensors for the
Detection of Cutaneous Leishmaniasis Disease in Exhaled Breath
Tesfalem Geremariam Welearegay, Mohamed Fethi Diouani, Lars Österlund,
Florina Ionescu, Kamel Belgacem, Hanen Smadhi, Samira Khaled,
Abdelhamid Kidar, Umut Cindemir, Dhafer Laouini, and Radu Ionescu
ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b00759 • Publication Date (Web): 07 Nov 2018
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Page 1 of 35 ACS Sensors

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4 Ligand-capped Ultrapure Metal Nanoparticle Sensors for the
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7 Detection of Cutaneous Leishmaniasis Disease in Exhaled Breath
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13 Tesfalem Geremariam Welearegaya,,§,*, Mohamed Fethi Diouanib,§,*, Lars
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17 Österlundc,d,§, Florina Ionescua, Kamel Belgacemb, Hanen Smadhie, Samira
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20 Khaledf, Abdelhamid Kidarg, Umut Cindemirc,d, Dhafer Laouinih and Radu
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24 Ionescua,d,*
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30 a MINOS-EMaS,
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Department of Electronics, Electrical and Automatic Engineering,
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34 Rovira i Virgili University, Tarragona 43007, Spain
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b Institut Pasteur de Tunis, LR11IPT03, Laboratory of Epidemiology and Veterinary
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40 Microbiology (LEMV), University Tunis El Manar, Tunis-Belvédère 1002, Tunisia
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43 c Molecular Fingerprint AB Sweden, Uppsala 75655, Sweden
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46 d The Ångström Laboratory, Division of Solid State Physics, Department of Engineering
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50 Sciences, Uppsala University, Uppsala 75121, Sweden
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e Ibn Nafis Pneumology Department, Abderrahman Mami Hospital, Ariana 2080,
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56 Tunisia
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3 f Parasitology-Mycology Laboratory, Charles Nicolle Hospital, Rue 9 Avril 1938, Tunis
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7 1006, Tunisia
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g Regional Hospital Houssine Bouzaiene of Gafsa, Gafsa Douali 2100, Tunisia
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13 h Institut Pasteur de Tunis, LR11IPT02, Laboratory of Transmission, Control and
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16 Immunobiology of Infections (LTCII), University Tunis El Manar, Tunis-Belvédère 1002,
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Tunisia
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§ These authors contributed equally to this work
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27 * Corresponding author. Tel: +34 977 25 65 72; fax: +34 977 55 96 05
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31 Email: tesfalemgeremariam.welearegay@urv.cat; radu.ionescu@urv.cat;
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34 fethi.diouani@gmail.com
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39 Abstract
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42 Human cutaneous leishmaniasis, although designated as one of the most neglected
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44 tropical diseases, remains underestimated due to its misdiagnosis. The diagnosis is
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mainly based on the microscopic detection of amastigote forms, isolation of the parasite,
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49 or the detection of Leishmania DNA, in addition to its differential clinical
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51 characterization; these tools are not always available in routine daily practice, and they
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54 are expensive and time consuming. Here we present a simple-to-use, non-invasive
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57 approach for human cutaneous leishmaniasis diagnosis, which is based on the
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4 analysis of volatile organic compounds in exhaled breath with an array of
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7 specifically-designed chemical gas sensors. The study was realized on a group
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11 of n = 28 volunteers diagnosed with human cutaneous leishmaniasis and a group
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14 of n = 32 healthy controls, recruited in various sites from Tunisia, an endemic
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country of the disease. The classification success rate of human cutaneous
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21 leishmaniasis patients achieved by our sensors test was 98.2% accuracy, 96.4%
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24 sensitivity, and 100% specificity. Remarkably, one of the sensors, based on CuNPs
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functionalized with 2-mercaptobenzoxazole, yielded 100% accuracy, 100% sensitivity,
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29 and 100% specificity for human cutaneous leishmaniasis discrimination. While AuNPs
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31 have been the most extensively used in metal nanoparticles-ligand sensing films for
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33 breath sensing, our results demonstrate that chemical sensors based on ligand-capped
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36 CuNPs hold also great potential for breath volatile organic compounds detection.
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38 Additionally, the chemical analysis of the breath samples with gas chromatography
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40 coupled to mass spectrometry identified nine putative breath biomarkers for human
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43 cutaneous leishmaniasis.
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Keywords: Chemical gas sensors; Metal nanoparticles – ligand
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51 nanoassemblies; Exhaled breath analysis; Diagnosis; Human cutaneous
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54 leishmaniasis; Biomarkers
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3 Cutaneous Leishmaniasis (CL) is a complexe disease caused by infection with different
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species of Leishmania (L.) protozoan parasite, which is transmitted by the bite of a female
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8 sandfly, and causes different clinical manifestations in humans.1-2 Despite huge efforts to
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10 minimize their invasiveness and control their spread, these parasites still circulate in 98
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countries, where they have affected 12 million people, induce 1.5 million cases per year,
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15 and maintain 397 million people at risk. Ecological and environmental changes,
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17 population displacements, civil wars, mobilisation of water resources, and establishment
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19 of new irrigated zones through newly built dams, in addition to the absence of an effective
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22 vaccine and drug-resistant populations, explain their persistence and endemicity.
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24 Human CL (HCL) is endemic in Central and South America, Africa, Asia and Southern
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28 Europe, and is expressed either as self-healing sores or as more chronic or
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31 disseminated muco-cutaneous lesions depending on the causative species.
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35 Severe forms constitute a major public health problem in terms of case
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38 management and treatment, disability-adjusted life years (DALYs) lost and
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42 economic productivity reduction, in addition to the social stigma associated with
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45 deformities and disfiguring scars.2
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48 In Tunisia, HCL caused by Leishmania (L.) major parasites affects urban and suburban
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51 areas in the center and south of the country, where the disease is endemo-epidemic. In
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53 most cases, infections by parasitic leishmania remain asymptomatic,3-4 and is rather a
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4 benign disease manifested most frequently as single or multiple crusted
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7 cutaneous ulcers developing at the site of parasite inoculation.
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10 HCL due to L. major is classically diagnosed based on clinical features, microscopic
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examination of skin-scraping smear, Polymerase Chain Reaction (PCR), and isolation
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15 and molecular identification of the parasite. The later ones are not always available in
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17 routine daily practice and are time consuming, while the former one depends on the host’s
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immunity, parasite species and differential diagnoses. In addition, none of them showed
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22 an adequate sensitivity and accuracy for the diagnosis and detection of HCL disease at
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24 the early stage of infection.
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26 Early detection and prompt treatment might reduce HCL spread and avoid increases in
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29 morbidity and drug resistance. In this endeavor, a simple-to-use, rapid, and non-invasive
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31 diagnostic through exhaled breath analysis could be highly beneficial.
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33 The analysis of volatile organic compounds (VOCs) in human breath has been
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increasingly studied, and it holds a great promise for the development of non-invasive
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38 diagnostic tools.5-7 Given that breath sampling is simple and can be conveniently
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40 repeated, breath analysis has emerged as a potential emerging diagnostic approach for
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diseases detection and monitoring compared to other fluid-based diagnosis.8-10 Although
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45 several techniques have been employed for breath analysis,11 several challenges related
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47 to the identification of the diseases specific biomarkers as well as the lack of
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49 understanding how the detected VOCs in breath can be associated to the pathobiology
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52 and pathophysiology of the disease, still remain not completely elucidated. However,
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54 detection of a breath VOCs pattern by means of cross-reactive chemical gas sensors
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56 arrays combined with pattern recognition algorithms (i.e., “electronic nose” systems),
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holds the potential to tackle these challenges by utilizing the response of individual
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3 sensors to obtain a unique breath print profile of the disease. VOCs analysis in exhaled
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breath employing nanomaterial based gas sensors arrays has demonstrated remarkable
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8 progress in the detection and diagnosis of different diseases, including diabetes,12 renal
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10 disease,13 gastric cancer,14 Alzheimer and Parkinson disease,15 asthma and obstructive
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pulmonary disease,16-17 lung cancer,18-22 and a number of other diseases.23
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15 Chemiresistive gas sensors based on molecularly modified metal nanoparticles (MNPs)
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17 have been successfully employed for breath analysis and the diagnosis and monitoring of
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19 several diseases.24-29 This could be related to their broad cross reactive capability to
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22 multicomponent exhaled breath VOCs provided by the broad range of organic ligands
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24 that can be attached to the MNPs, as well as because they can be conveniently operated
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Here we present, to the best of our knowledge, the first study regarding the diagnosis of
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31 HCL by analyzing the VOCs profile of patients breath with an array of chemical gas
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33 sensors based on different ligand-capped ultrapure metal nanoparticles.
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Complementarily, the breath biomarkers of HCL were identified by analytical studies,
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38 and their possible involvement in the pathogenesis of HCL is discussed.
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41 Experimental Section
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45 Study population and breath samples collection
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48 Volunteers diagnosed with cutaneous leishmaniasis (HCL; n = 28) and healthy controls
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50 (CCL; n = 32), aged over 18 years old, were recruited at Tunis Charles Nicolle University
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52 Hospital (North East Tunisia), Ben Arous Regional Hospital (North East Tunisia),
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55 Abderrahman Mami University Hospital (Ariana, North East Tunisia) and Gafsa
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57 Houssine Bouzaiene Regional Hospital (South East Tunisia). Patients’ diagnosis was
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59 based on microscopic tests (examination of skin-scraping smear samples) and clinical
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3 aspects of the lesions. The controls were selected from the medical staff of these clinics.
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The group of control volunteers was age and sex matched with the CL patients, as much
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8 as possible. The study population is presented in the Supporting Information.
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10 The breath samples of both patients and controls were acquired in the same room of the
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hospitals were the patients were attended, after fasting overnight. Exhaled breath samples
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15 of volunteers were collected with BioVOCTM breath samplers (Markes International,
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17 UK), as explained in Supporting Information.
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20 Chemical gas sensors
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23 Ultrapure gold nanoparticles (AuNPs), copper nanoparticles (CuNPs) and platinum
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25 nanoparticles (PtNPs) were fabricated and deposited on sensing substrates employing an
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27 advanced gas deposition (AGD) technique (Figure 1), as explained in detail elsewhere.30-
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Briefly, a piece of pure metal precursor was placed in a carbon crucible inside an
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32 induction coil in the evaporation chamber of the AGD setup. The metal was melted by
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34 inductively heating the crucible by applying a suitable electrical power to the induction
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coil. The metal atoms evaporated from the surface of the melted metal piece (either Au,
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39 Cu, or Pt) were cooled under a laminar flow of high-purity inert He gas introduced from
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41 beneath the metal piece, leading to the formation of tiny nuclei in the nucleation zone
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43 above the melt, which subsequently formed metal nanoparticles by collision in the gas
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46 flow. p-doped Si sensing substrates with two parallel, 15 µm gapped, gold electrodes
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48 patterned on top, were mounted on a movable stage placed inside the upper (deposition)
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50 AGD chamber, which was kept at a lower pressure compared to the evaporation AGD
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chamber. The particles generated in the lower chamber were accelerated through a 3 mm
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55 transfer pipe to the upper chamber because of the pressure difference between the two
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57 chambers, resulting in the deposition of ultrapure metal nanoparticles when impacting
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the sensing substrate. The experimental conditions for each kind of MNPs fabricated are
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3 presented in the Supporting Information. The MNPs mean grain size was ~ 10 nm for
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AuNPs, ~ 3 nm for PtNPs, and ~ 4 nm for CuNPs, as determined from XRD analysis.31
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Figure 1. Schematic representation of the AGD setup.
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35 Surface functionalization of the as-deposited metal nanoparticles was performed by dip
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37 coating the sensing devices into ligand solutions.30-31 Several types of ligands were
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39 employed: 1-dodecanethiol, 11-mercaptoundecanol, 2-mercaptobenzoxazole, 11-
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42 mercaptoundecanoic acid, which were dissolved in 20 mL ethanol. The list of metal
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44 nanoparticles – ligand nanoassemblies used as sensing material for the sensors array
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46 developed for this study is presented in Table 1. Importantly, the mean grain size of the
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MNPs was not altered by MNPs functionalization with the organic ligands.30
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52 Table 1. List of chemical sensors based on MNPs-ligand nanoassemblies.
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55 Sensor Metal Nanoparticles Molecular organic Resistance
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3 S1 CuNPs 4 2-mercaptobenzoxazole
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7 S2 CuNPs 4 11-mercaptoundecanol 2.3
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10 S3 AuNPs 10 2-mercaptobenzoxazole
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14 S4 PtNPs 3 1-dodecanethiol 7.8
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17 S5 AuNPs 10 1-dodecanethiol
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21 S6 CuNPs 4 11-mercaptoundecanoic 4.5
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29 Sensing measurements
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31 The six chemical gas sensors were placed in a stainless steel test chamber (70×35×30
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33 mm3) provided with an inlet for introducing breath samples, and an outlet for exhausting
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the sample after measurement. The outlet opening was also used for realizing vacuum
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38 inside the test chamber prior to any new measurement by means of an electrical pump
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40 (Model C5, Electro A.D., Spain).
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42 For measuring the breath VOCs stored inside the storage tubes, the Tenax TA sorbent
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45 material was transferred into a 20 mL vial with a lid provided with a septum, which was
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47 hermetically sealed. The vial was heated for 10 min at 275 ºC in a conventional oven
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49 (UNE 300, Memmert, GmbH, Germany), which released the VOCs trapped by the Tenax
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52 sorbent material that formed a headspace inside the sealed vial. A volume of 10 mL of
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54 the headspace was captured and injected into the sensors test chamber through the inlet
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56 opening septum by means of a gas tight syringe (VWR International EuroLab, Spain).
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3 Each breath sample measurement comprised the following cycles: 320 sec under low
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vacuum conditions (~ 650 mbar), followed by 320 sec exposure to the breath VOCs under
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8 stationary conditions (pressure inside sensors chamber: ~ 875 mbar ± 3%), and by 30 min
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10 flushing with synthetic dry air (2 L/min flow rate) for sample evacuation and cleaning
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sensors surface and the measurement chamber. During the vacuum stage and breath
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15 sample exposure, the six sensors were sequentially operated at 8 V for 10 sec in a
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17 switching mode, followed by 70 sec of inactivity, and the dc current through the sensors
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19 was acquired for further analysis. During the inactivity of one of the sensors, all the other
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22 sensors were operated each one for 10 sec in the mentioned switching mode. Each
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24 measurement cycle comprised furthermore 20 sec of inactivity for all sensors, which
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26 totally provided 320 sec for the four measurement cycles realized. For speeding up the
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cleaning process after sample measurement, the sensors were operated at 10 V for 10 sec
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31 followed by 70 sec of inactivity in the forementioned switching mode. A high precision
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33 power source (B2902A, Keysight Technologies, Hungary) and a high resolution data
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acquisition system (34992A LXI/Data Acquisition Keysight Technolgies, Hungary) were
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38 employed for delivering the desired operating voltage to the sensors and for acquiring
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40 sensors data, respectively. Sensors operation was controlled with a custom made
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42 LabView 2014 program.
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46 Analytical measurements
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48 A 7890A gas chromatograph (GC) coupled to a 7200 quadruple time of flight (Q-TOF)
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50 mass spectrometer (Agilent Technologies, Santa Clara, CA, USA), was employed for the
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chemical analysis of the breath samples. For this end, the Tenax TA material storing the
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55 breath volatiles was transferred into a 20 mL glass vial that was sealed with a septum and
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57 placed for 20 min in an oil bath heated at 100 ºC on a temperature-controlled hotplate.
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This procedure caused the VOCs trapped by the sorbent material to desorb and form a
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3 headspace above the Tenax material inside the sealed vial. Solid Phase Micro-Extraction
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(SPME) with Divinilbenzene/Carboxene/Polydimethylsiloxane (DVB/CAR/PDMS)
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8 fiber, introduced for 30 min in the headspace formed by the released breath volatiles
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10 inside the vial, was used to capture and concentrate the VOCs and to inject them into the
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GC/Q-TOF splitless port for analysis. The oven conditions of the chromatograph were:
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15 desorption time: 1 min, desorption temperature: 250 ºC. The oven program was set as
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17 follows: initial temperature: 50 ºC, 10 ºC/min ramp until 155 ºC, 20 ºC/min ramp until
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19 270 ºC, 10 min at 270 °C. Mass spectrometry measurements were performed by electron
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22 impact ionization, with 70 eV electron energy and the temperature source held at 230 ºC.
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24 Mass-to-charge ratios between 35 and 400 m/z were acquired at 5 spectra/s rate.
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26 Chromatograms deconvolution was processed employing the Unknown Analysis
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software (Qualitative and Quantitative Analysis B.07.00, Unknown Analysis, Agilent
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31 Technologies, Santa Clara, USA) operated in the automatic mode, with a match factor
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33 set to 70. Compounds identification was achieved using the NIST 14 mass spectral
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library.
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39 Data analysis
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41 Sensors signals (electrical dc current acquired through the sensors during breath samples
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43 exposure) were initially scaled to zero-mean and unitary variance for each sample
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46 measured. Three features were extracted from each sensor response to a breath sample:
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48 F1: Mean dc current acquired during 1.22 seconds (11 sampling points) at the middle of
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50 sensor exposure to the breath sample; F2: Mean dc current acquired during the last 1.22
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seconds (last 11 sampling points) of sensor exposure to the breath sample; F3: Area under
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55 the response curve during the last 1.78 seconds (last 16 sampling points) of sensor
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57 exposure to the breath sample. The schematic representation of the features extracted is
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provided in Supporting Information These features were introduced as input to two
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3 different pattern recognition algorithms, Principal Component Analysis (PCA) and
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Discriminant Factor Analysis (DFA), for building classification models for the HCL and
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8 CCL groups. PCA is a linear and unsupervised technique that reduces multidimensional
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10 data by calculating new orthogonal variables, called principal components (PC), as a
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linear combination of the input variables, which capture the maximum variance in the
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15 data, for projecting them into a low dimensional space.33 DFA is a linear and supervised
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17 method that calculates new orthogonal variables called canonical variables (CV) as a
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19 linear combination of the input features in such a way to minimize the variance within
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22 each group and maximize the variance between groups.33-34 The classification success
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24 rate of the models built was estimated based on samples grouping for PCA,35 and through
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26 leave-one-out cross-validation for DFA for avoiding unrealistic results because of
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overtraining. The accuracy, sensitivity, and specificity of HCL classification were
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31 calculated based on true positives (TP), true negatives (TN), false positives (FP) and false
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33 negatives (FN) rates, as follows: accuracy = (TP+TN)/(TP+TN+FP+FN); sensitivity =
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TP/(TP+FN); specificity = TN/(TN+FP).
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38 The two-tailed statistical t-test for two samples assuming unequal variance was applied
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40 for biomarkers identification in the GC-MS analysis, using a standard cut-off value α =
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42 0.05 for estimating significant variance between HCL and CCL groups. DFA
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45 classification models were calculated with the identified biomarkers, and HCL
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47 classification accuracy, sensitivity and specificity were estimated through leave-one-out
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49 cross-validation.
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The diagnostic models for the sensors were further evaluated using the receiver operating
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54 characteristic (ROC) curve analysis of the first principal component (PC1) of the PCA
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56 model that captures the highest variance in the data, and with the first and most
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discriminating canonical variable (CV1) of the DFA model, respectively. The ROC curve
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3 is plotted as a function of true positive rate (sensitivity) vs false positive rate (1-
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specificity) at different cut-off points, and the area under the curve (AUC), defined as the
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8 area of the ROC curve minus the random results, reflects how well the diagnostic test
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10 (accuracy) distinguishes patients from controls.
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Different confounding factors were assessed for both the sensors test and the GC-MS
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15 analysis for disregarding the effect of external conditions on the results obtained:
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17 volunteers’ gender (male/female) and age (divided here in three groups: young (18-29
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19 years), middle-aged (30-49 years), and senior (> 50 years)). Smoking habit was not
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22 assessed as a confounding factor because of the low number of smoking volunteers that
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24 participated in this study.
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Results and Discussion
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32 Exhaled breath analysis with the chemical sensors array
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35 Breath samples collected from 56 volunteers (28 HCL patients and 28 CCL controls)
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37 were measured with the chemical sensors array (the other four CCL samples were
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40 employed to realize preliminary measurements for setting-up the measurement system).
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42 As a representative example, Figure 2a shows the response of sensor S1 under vacuum
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44 conditions, and to the breath samples of HCL and CCL volunteers, during four operation
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cycles, illustrating the rapid and reproducible response of the sensor towards the different
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49 samples analyzed.
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51 The MNPs are electrically insulated as deposited, but upon ligand functionalization they
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become conductive, albeit with an intrinsic high resistance (Table 1). The organic ligand
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56 provides sorption sites for the breath VOCs, and the conductance of the sensing layer is
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58 modified upon VOC adsorption on the ligands.24, 29-30, 36-37 The decrease in the electrical
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3 VOCs sorption onto the MNPs-ligand matrix favoring film swelling, and hence
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increasing the distance between the MNPs cores, which thereby reduces the electron
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8 transfer between the MNPs.24, 36, 38 The swelling effect is reversible when the sample is
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10 evacuated from the sensors test chamber,36, 39-40 leading to the observed recovery of
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sensor’s baseline conditions.
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15 On the other hand, it is noted that the sensor response presents different profiles to the
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17 breath VOCs of HCL and CCL volunteers, suggesting that it is possible to discriminate
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19 breath volatiles patterns of the HCL and CCL groups. To assess the sensor’s capability
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22 to discriminate the two study groups (HCL and CCL), an unsupervised PCA pattern
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24 recognition algorithm was computed with the three features extracted from sensor’s
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26 response to the breath samples of all volunteers from this study. Figure 2b presents the
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patients classification achieved by the PCA plot. The first principal component (PC1) of
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31 the PCA model, which accounted for 99.8% of the variance in the data (Figure 2c),
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33 provided 80.3% accuracy, 78.6% sensitivity and 82.1% specificity for HCL
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discrimination, while the AUC of the ROC curve (Figure 2d), indicated 87.6% accuracy.
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38 Given these promising results, the supervised DFA pattern recognition algorithm was
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40 also investigated for training the discrimination model between the HCL and CCL
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42 groups. Figure 2e shows the classification obtained by the first canonical variable (CV1)
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45 of the DFA model built with the three features extracted from sensor’s response to the
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47 breath samples of all volunteers, where all volunteers were correctly classified in function
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49 of their health state. The HCL classification accuracy, sensitivity and specificity
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calculated through leave-one-out cross-validation were all 100%. The diagnostic model,
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54 further validated employing the ROC curve analysis (Figure 2f), gave AUC equal to
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56 1.00, corresponding to 100% accuracy.
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49 Figure 2. (a) Typical response of one of the sensors (sensor S1, based on CuNPs
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51 functionalized with 2-mercaptobenzoxazole) towards exposures to vacuum and breath
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53 samples of HCL and CCL volunteers; (b) PCA plot computed with the features extracted
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56 from the response of sensor S1 to the breath samples of all volunteers; each volunteer is
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58 represented by one point in the plot; (c) Boxplot of the first principal component (PC1)
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3 S1 to the breath samples of all volunteers; each volunteer is represented by one point in
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the plot; the standard deviation of the PC1 values is represented by the error bars, while
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8 the boxes represent the 95% confidence interval; the dashed line represents the threshold
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10 classification line between the two groups, HCL patients being estimated below the
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12
dashed line and healthy controls above the dashed line; (d) ROC curve analysis of the
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15 PC1 values of the PCA model; (e) Boxplot of the most discriminative canonical variable
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17 (CV1) of the DFA model computed with the features extracted from the responses of
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19 sensor S1 to the breath samples of all volunteers; each volunteer is represented by one
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21
22 point in the plot; the standard deviation of the CV1 values is represented by the error
23
24 bars, while the boxes represent the 95% confidence interval; the dashed line represents
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26 the threshold classification line between the two groups, HCL patients being estimated
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above the dashed line and healthy controls below the dashed line; (f) ROC curve analysis
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31 of the CV1 values of the DFA model.
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34 Pursuing the same analysis for the other chemical sensors (S2-S6), the results presented
35
36 in Table 2 were obtained. The different sensors comprise different MNP-ligand
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38
39 nanoassemblies, which differ in MNPs core size, molecular ligand structure,
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41 nanoassembly morphology and surface coverage.31 It is clear from the results collected
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43 in Table 2 that sensors based on CuNPs (i.e., S1, S2, and S6), provided the highest
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45
46
classification accuracy. While AuNPs have been the most extensively used in MNPs-
47
48 ligand sensing films for breath VOCs sensing,19, 23, 25-26 our results demonstrate that
49
50 chemical sensors based on ligand-capped CuNPs hold great potential for breath VOC
51
52 detection. The sensing properties of MNPs-ligand nanoassemblies depend on a number
53
54
55 of factors, among which the MNPs core size has been assumed to be of paramount
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57 importance, and determines the effectiveness of film swelling and dielectric changes
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59 upon VOCs sorption.41 It has further been reported that the electronic conduction of the
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3 nanoassembly, and hence its sensitivity to VOCs, depends on the morphology and size
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of the MNPs cores as well as on how the ligand is attached to the metal cores.40-42 Thus,
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8 the high sensing capability achieved in the present study by the CuNPs-ligand
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10 nanoassemblies might be attributed to the small CuNPs core size (~ 4 nm), ligand
11
12
attachment and film morphology, which exhibited a well-ordered structure in the case of
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15 the CuNPs capped with 2-mercaptobenzoxazole, as we have previously shown.31 As a
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17 result, the swelling efficiency of the film might be predominantly governed by the
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19 formation of percolation pathways upon VOCs adsorption,27, 40 maximizing electrons
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21
22 transfer.
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24 The overall classification ability of the chemical sensors array was next considered for
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26 assessing the synergic effect of an array of non-specific cross-reactive sensors to respond
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29
to a variety of VOCs in the exhaled breath, enabling the identification of specific breath
30
31 print patterns in multicomponent VOCs mixtures.17, 19 Employing this approach, the DFA
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33 model built with the three features extracted from the responses of all six sensors from
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35
the array to the breath samples analyzed yielded 98.2% accuracy, 96.4% sensitivity and
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38 100% specificity in the classification of HCL patients (last column in Table 2). Thus,
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40
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42 the sensor array achieved excellent discrimination capability between the study
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45 groups, suggesting that the cross reactive sensors array can be devised to
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49 achieve distinct and robust sensing features for multicomponent VOCs pattern
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51
52 analysis. This is because the combination of different sensors, with different
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MNPs and functional organic ligands, provide the desired panels of MNPs-
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4 ligands for combined sensitivity and selectivity towards the disease specific
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7 breath VOCs pattern.
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10 The role of confounding factors (gender and age in the case of our study), which could
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influence sensors performance, was finally assessed for all the discriminative models
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15 built in this study. The results of these analyses confirmed that the obtained HCL
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17 classification results were not affected by patients age, but could be slightly affected by
18
19
patients gender (the accuracy of age classification ranged between 26.8% and 55.4% for
20
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22 the different models, while the accuracy of gender classification ranged between 37.5%
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24 and 60.7%).
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27 Table 2. HCL classification results obtained by the DFA models built with the
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29 responses of each individual sensor, as well as with the overall responses of all
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31
sensors from the array.
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34
35 S1 S2 S3 S4 S5 S6 Sensors array
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39 Accuracy 100 80.4 73.2 73.2 78.6 85.7 98.2
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42 Sensitivity 100 78.6 75 57.1 75 89.3 96.4
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Specificity 100 82.1 71.4 89.3 82.1 82.1 100
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49 TP/TN 28/28 22/23 21/20 16/25 21/23 25/23 27/28
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FP/FN 0/0 5/6 8/7 3/12 5/7 5/3 0/1
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59 Chemical analysis of the breath samples
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3 Breath samples of 28 HCL patients and 32 healthy controls were analyzed with the GC/Q-
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6
TOF system. Approximately 120 chemical compounds were identified in each breath
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8 sample. Among them, nine compounds were found in statistically different
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10 concentrations between HCL and CCL groups. They are indicated in Table 3 as putative
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12
breath biomarkers for HCL. All of them were present in at least 80% of the breath
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15 samples from each study group. Importantly, the abundance (mean area under the
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17 chromatographic peak) of all these compounds was higher in the breath of HCL patients
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19 as compared with the healthy controls (Figure 3a). Moreover, the mean variance of these
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22 compounds within the HCL group was much smaller than in the case of CCL group,
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24 indicating the homogeneity of the abundance of the HCL breath biomarkers in the breath
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26 of all patients. Although presenting lower abundance in the CCL group, the higher spread
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28
29
of these compounds in the controls group is due to the higher variability of the normal
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31 state.
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34 Table 3. Putative breath biomarkers for human cutaneous leishmaniasis identified
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36 in the present study
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38
39
40 VOCs Compounds CAS No m/z R.T p-value
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42 VOC 01 2,2,4-trimethyl pentane 540-84-1 57.05 4.35 0.028
43
VOC 02 4-methyl-2-ethyl-1-pentanol 106-67-2 67.05 4.9 0.042
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45 VOC 03 Methylvinyl ketone 78-94-4 43.05 5.28 0.015
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47 VOC 04 Nonane 1184-2 43.05 5.99 0.02
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49 VOC 05 2,3,5-trimethyl hexane 1069-53-0 85.01 6.53 0.012
50 VOC 06 Hydroxy-2,4,6-trimethyl-5-(3- 1139-17-9 81.06 9.50 0.001
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52 methyl-2 butenyl) cyclohexyl)
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54 methylacetate
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VOC 07 Octane 111-65-9 57.05 9.75 0.008
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57 VOC 08 3-ethyl-3-methylheptane 17302-01-1 71.06 10.08 0.01
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59 VOC 09 2-methyl-6-methylene-octa-1,7- 22459-10-5 67.05 10.86 0.03
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3 dien-3-ol
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7 The diagnostic potential of the breath biomarkers to discriminate between HCL and CCL
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9 was assessed by building classification models based on the DFA algorithm using as
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11 input different biomarkers combinations. The DFA classification model built with the
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13
14
abundances of only two biomarkers (VOC03 and VOC06) yielded 80% accuracy, 71.4%
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16 sensitivity and 87.5% specificity, while the DFA classification model built with the
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18 abundances of three biomarkers (VOC01, VOC06 and VOC07) yielded 80% accuracy,
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75% sensitivity and 84.5% specificity (Figure 3b), revealing the remarkable
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23 discrimination capability of the breath biomarkers between the study groups.
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38 Figure 3 (a) Abundance (mean area under the chromatographic peak) of the HCL breath
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40 biomarkers; (*) statistically significant difference (p < 0.05) between HCL and CCL
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43
groups; (**) statistically significant difference (p < 0.01) between HCL and CCL groups;
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45 (b) Boxplot of the first canonical variable (CV1) of the DFA model built with the
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47 abundances of three biomarkers (VOC01, VOC06, VOC07); each volunteer is
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49 represented by one point in the box plot; the standard deviation of the CV1 values is
50
51
52 represented by the error bars, while the boxes represent the 95% confidence interval; the
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54 dashed line represents the threshold classification line between the two groups, HCL
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56 patients being estimated above the dashed line and healthy controls below the dashed
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3 line; (c) Biomarkers correlation map; (+1), (-1) and (0) indicate maximum positive,
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maximum negative, and no correlation, respectively.
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9 However, not all VOC biomarkers are equally significant for distinguishing between the
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11 diseased and healthy states. This is evidenced by the correlation matrix of biomarkers
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13
abundances (Figure 3c), which indicates that several biomarkers are highly correlated
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16 with each other (VOC03, VOC04 and VOC05), while others (VOC02 and VOC06)
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18 showed a negative correlation with the first ones. On the other hand, one biomarker
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20 (VOC07) showed a general correlation with almost all VOCs (with the exception of
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23 VOC02 and VOC06), while VOC08 and VOC09 exhibited positive correlation with all
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25 the other biomarkers. This explains why the DFA model built with the abundances of all
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27 biomarkers presented a lower classification potential (66.7% accuracy) than the models
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30
built with a limited number of selected biomarkers. An attempt to elucidate the
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32 biochemical processes that originated the biomarkers identified in the present study is
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34 presented below.
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36
First signs of lesions due to HCL, a widely distributed vector-borne disease that is never
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39 fatal, even if non-treated, are characterized by the appearance of a small erythema at the
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41 site of a sandfly bite. This erythema develops into a papule and nodule and the lesion
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43 becomes ulcerated. Both innate and adaptive immune systems form together the driving
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46 forces, which shape the disease outcome. The initial interaction with the innate immune
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48 cells shapes the subsequent effector response. Effector function of activated CD4+ T
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50 helper cells is an integral component in determining the course of the disease. A Th1-like
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53
response activates macrophages to clear the parasite. The cascade induced by interferon
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55 gamma (IFN-γ) in infected macrophages results in the production of Reactive Oxygen
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57 Species (ROS) that have the ability to kill intracellular Leishmania. In addition to ROS,
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IFN- γ and tumor necrosis factor (TNF) synergistically induce Nitric Oxide Synthase
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3 (iNOS) in macrophages, and Nitric Oxide (NO) production further promotes effective
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parasite killing. The increased IFN-γ production to elevate ROS level for controlling
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8 parasite invasion is associated with increased inflammatory reaction and development of
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10 cutaneous ulcers in the skin.43 The imbalance of the general equilibrium between the
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12
formation and deactivation of ROS and free radicals can boost oxidative stress in the
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15 body. Thus, ROS can damage cellular and subcellular structures such as lipids, and
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17 induce upregulation of cytochrome p-450 enzymes to catalyze the oxidation of organic
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20 chemicals in the human tissues.44 More specifically, lipid peroxidation occurs as a
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23 result of oxidative degradation of lipids, when free radicals attach themselves to
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27 electrons in cell membranes and thereby damage components of the cell
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30 membrane.45 Due to the presence of reactive hydrogen of the methylene groups
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34 between the multiple double bonds, polyunsaturated fatty acids (PUFAs) are
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37 commonly affecting the cell components.46-48 For example, aldehyde, ethane,
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41 pentane, malondialdehyde, acrolein, hydroxynonenal and crotonaldehyde are
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44 well known PUFA products in the lipid peroxidation process.47
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47 Nevertheless, the origin of the exhaled breath VOCs that were putatively identified in the
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50 breath of HCL patients is not completely elucidated. Although several biochemical
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53 pathways are possible, the peroxidation of PUFAs during the pathophysiology of
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4 HCL might be considered as the main source of aliphatic hydrocarbons in the
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7 exhaled breath, such as nonane (VOC04) and octane (VOC07).
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9
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11 Earlier reports showed that enhanced levels of ethane and pentane can be
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14 associated to lipid peroxidation during oxidative stress in critical body
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conditions.49-50 The generation of branched alkanes such as 2,2,4-
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21 trimethylpentane (VOC01), 2,3,5-trimethylhexane (VOC05) and 3-ethyl-3-
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23
24
25
methylpentane (VOC08) might be also related to the byproducts of PUFA
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28 peroxidation. Nevertheless, it is argued that oxidative stress mechanism in
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mammals cannot produce straight chained or branched alkanes, and hence such
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35 branching alkanes occur during the isoprenoid synthesis.51 On the other hand,
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38
alkanes are also present in the environment and are inhaled on a daily basis,
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40
41
42 being broken in the liver by the cytochrome p-450 enzymes, therefore the
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45
exogenous origin of these biomarkers cannot be discarded.
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49 Alcohols such as 4-methyl-2-ethyl-1-pentanol (VOC02) and 2-methyl-6-
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52 methylene-octa-1, 7-dien-3-ol (VOC09) might originate from the hydrocarbon
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55
56 metabolism. This could be either due to the alcohol metabolism catalyzed by
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59 cytochrome p-450 enzymes in the liver, or due to the oxidation of several different
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4 alcohols by alcohol dehydrogenase in the body.52 Moreover, byproducts of lipid
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7 peroxidation might underway oxidation when they are catalyzed by the peripheral
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9
10
11 cell enzymes, being then exhaled through breath.
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15 Ketones such as methylvinyl ketone (VOC03) are related to excessive fatty acid
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18 oxidation due to metabolic changes produced by HLC disease onset, when the
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20
21
22 body uses fat instead of glucose for energy. For instance, acetone is produced
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25 in the liver by decarboxylation of acetoacetate during lipolysis as a secondary
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27
28
29 source of energy for the body.47 Thus, methylvinyl ketone might be produced as
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31
32 a result of secondary metabolites in lipid peroxidation. Hydroxy-2, 4, 6-trimethyl-
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34
35
36 5-(3-methyl-2-buytenyl)cyclohexylmethylacetate (VOC06) belongs to the
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38
39 branched ketone group and might be also related to excessive oxidation of fatty
40
41
42
43 acids.
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45
46 Although deeper investigations are required to fully understand the origin of the
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48
49
50 HCL breath biomarkers identified in this study, based on the above analysis we
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53 associate them with biochemical changes linked to molecular processes such as
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57 oxidative stress, lipid metabolism, cytochrome p-450 enzymes, and carbohydrate
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4 metabolisms, produced in the body during pathogen infections with leishmania
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7 parasite.
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9
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11 The remarkable results obtained by the sensors test can be attributed to chemical
12
13
14 and physical affinity features of the cross-reactive sensors from the array to
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16
17
18
specific families of compounds. The molecular ligands of sensors S1, S2, S3,
19
20
21 and S6, with polar and aromatic functional groups in the tail, are likely to have
22
23
24
25
good affinity to breath VOCs with similar functional characteristics, such as
26
27
28 methylvinyl ketone, 4-methyl-2-ethyl-1-pentanol, hydroxyl-2,4,6-trimethyl-5-(3-
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30
31
32
methyl-2-butenyl)cyclohexyl methylacetate and 2-methyl-6-methylene-octa1,7-
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34
35 dien-3-ol. On the other hand, the non-polar tail of the ligand of sensors S4 and
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38
39
S5 interacts more preferably with non-polar VOCs such as 2,2,4-
40
41
42 trimethylpentane, nonane, octane, 2,3,5-trimethylhexane, and 3-ethyl-3-
43
44
45
methylpentane.
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47
48
49 Although the desorption temperature employed in our study for the GC/Q-TOF
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51
52 studies and sensors measurements were different and possibly influenced the
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55
56 number of breath VOCs desorbed from the Tenax material, which could limit the
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59 number of putative HCL breath biomarkers identified, it is important to note that
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4 these are independent and complementary techniques. The GC/Q-TOF studies
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7 identify the specific compounds that compose the sample analyzed, while the
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9
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11 chemical gas sensors are not compound selective; they respond to the collective
12
13
14 volatiles sample and, depending on the sensing material employed, they are
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16
17
18
more sensitive to some classes of VOCs. The panels of VOCs identified by
19
20
21 GC/Q-TOF did however form a sufficient basis for a rational design of the sensors
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23
24
25
array for HCL diagnosis.
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27
28
29 Conclusions
30
31
32 To the best of our knowledge, this is the first ever report on exhaled breath analysis as a
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34 way to non-invasively diagnose human cutaneous leishmaniasis with chemical sensors.
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36
37
In particular, we show that a purposefully designed chemical sensors array can be trained
38
39 for the accurate classification of the breath prints of patients with cutaneous
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41 leishmaniasis, achieving 98.2% accuracy, 96.4% sensitivity and 100% specificity in the
42
43
classification of HCL patients. Remarkably, CuNP-based nanoassemblies yields higher
44
45
46 accuracy than AuNPs sensors, which up to date by far have been the most used biosensor
47
48 MNP material in breath detection Also remarkably, the CuNPs functionalized with 2-
49
50 mercaptobenzoxazole nanoassembly sensor yielded 100% accuracy, sensitivity and
51
52
53 specificity for HCL discrimination. The sensing methodology that we introduce in this
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55 paper is simple to use and interpret, and is amenable for use in both specialist and non-
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57 specialist settings. Therefore, it could provide a powerful tool for the facile, rapid and
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3 non-invasive diagnosis of HCL at an early stage, and would be especially important in
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6
regions lacking specialized healthcare diagnostic facilities.
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8 Additionally, we identified nine putative breath biomarkers for HCL, which presented
9
10 statistically different abundances in the breath of HCL patients with regard to the healthy
11
12
controls. It is important to remark here the homogeneity of the abundances of these
13
14
15 biomarkers in the cohort of 28 HCL patients that volunteered for this study. We discuss
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17 the possible origin of these biomarkers in light of previous reports related with cutaneous
18
19 diseases. Further investigation of their origin could provide new insight in the metabolism
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21
22 and biochemical pathway of HCL in patients’ body, which presumably could lead to the
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24 development of improved therapeutic strategies.
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26
27 Future studies will need to be conducted to elucidate the similarities and
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30 differences between HCL breath volatiles pattern and other cutaneous diseases.
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34
References
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4 Acknowledgments
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7 T.G.W acknowledges FPI predoctoral fellowship from MINECO (Spain). This
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11 project has received funding from the European Union’s Horizon 2020 research
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14 and innovation programme under the Marie Skłodowska-Curie grant agreement
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No. 645758.
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22 Conflict of Interest Disclosure
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25 The authors declare no competing financial interest.
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29 Supporting Information
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32 Supporting Information Available: The following file is available free of charge.
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35 Ethics statement. Study population. Breath samples collection. Experimental
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39 conditions for metal nanoparticles synthesis. Samples measurement approaches
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42 with the chemical gas sensors. Schematic representation of the sensors features
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