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Breathprinting and Early Diagnosis of Lung Cancer

Open ArchivePublished:March 08, 2018DOI:https://doi.org/10.1016/j.jtho.2018.02.026

      Abstract

      The electronic nose (e-nose) is a promising technology as a useful addition to the currently available modalities to achieve lung cancer diagnosis. The e-nose can assess the volatile organic compounds detected in the breath and derived from the cellular metabolism. Volatile organic compounds can be analyzed to identify the individual chemical elements as well as their pattern of expression to reproduce a sensorial combination similar to a fingerprint (breathprint). The e-nose can be used alone, mimicking the combinatorial selectivity of the human olfactory system, or as part of a multisensorial platform. This review analyzes the progress made by investigators interested in this technology as well as the perspectives for its future utilization.

      Keywords

      The electric nose (e-nose) reproduces the combinatorial selectivity appropriate to the human olfaction system. Unlike the human nose, gas mixtures are pumped towards the e-nose and, through a sampling device, are filtered to be then exposed to several sensors. In turn, these sensors produce signals which are processed through computerized algorithms based on pattern recognition and multivariate analysis.
      • Zou Y.
      • Wan H.
      • Zhang X.
      • Ha D.
      • Wang P.
      Electronic nose and electronic tongue.
      E-nose technology represents a promising innovation in the field of a “no touch” early diagnosis of lung cancer.
      • Shlomi D.
      • Abud M.
      • Liran O.
      • Bar J.
      • Gai-Mor N.
      • Ilouze M.
      • et al.
      Detection of lung cancer and EGFR mutation by electronic nose system.
      • Schumer E.M.
      • Trivedi J.R.
      • van Berkel V.
      • et al.
      High sensitivity for lung cancer detection using analysis of exhaled carbonyl compounds.
      In brief, the e-nose works by identifying fingerprints, or breathprints, defined by the activation of gas sensors exposed to volatile organic compounds (VOCs) that are markers of the cellular metabolism which are found in the exhaled breath but also in other organic fluids.
      • Shlomi D.
      • Abud M.
      • Liran O.
      • Bar J.
      • Gai-Mor N.
      • Ilouze M.
      • et al.
      Detection of lung cancer and EGFR mutation by electronic nose system.
      • Schumer E.M.
      • Trivedi J.R.
      • van Berkel V.
      • et al.
      High sensitivity for lung cancer detection using analysis of exhaled carbonyl compounds.
      Several investigators worldwide have already published the results of their preliminary studies yielding high accuracy for the e-nose in distinguishing between malignant and benign pulmonary nodules as well as the potential to identify an adenocarcinoma histotype and even EGFR status.
      • Shlomi D.
      • Abud M.
      • Liran O.
      • Bar J.
      • Gai-Mor N.
      • Ilouze M.
      • et al.
      Detection of lung cancer and EGFR mutation by electronic nose system.
      • Schumer E.M.
      • Trivedi J.R.
      • van Berkel V.
      • et al.
      High sensitivity for lung cancer detection using analysis of exhaled carbonyl compounds.

      The Historic Background of E-Nose Technology

      In 1971, Nobel Prize winner Pauling and his associates published what it is considered the first report showing by gas chromatography (GC) the presence of hundreds of chemical volatile compounds in human breath and urine.
      • Pauling L.
      • Robinson A.B.
      • Teranishi R.
      • Cary P.
      Quantitative analysis of urine vapour and breath by gas–liquid partition chromatography.
      Subsequent studies have identified several chemical derivatives of oxidative stress (Table 1) which can be produced by cancer-induced peroxidation of DNA bases.
      • Preti G.
      • Labows J.
      • Kostelic J.
      • Aldinger S.
      • Daniele R.
      Analysis of lung air from patients with bronchogenic carcinoma and controls using gas chromatography–mass spectrometry.
      • Phillips M.
      • Gleeson K.
      • Hughes J.M.B.
      • Greenberg J.
      • Cataneo R.N.
      • Baker L.
      Volatile organic compounds in breath as markers of lung cancer: a cross-sectional study.
      • Horvath I.
      • Lazar Z.
      • Gyulai N.
      • Kollai M.
      • Losonczy G.
      Exhaled biomarkers in lung cancer.
      • Poli D.
      • Carbognani P.
      • Corradi M.
      • Goldoni M.
      • Acampa O.
      • Balbi B.
      • et al.
      Exhaled breath volatile compounds in patients with non–small cell lung cancer: cross sectional and nested short-term follow-up study.
      Crucial to cancer development is the association of oxidative stress and the induction of cytochrome p-450 enzymes (CYP450), a group of oxidase enzymes (Fig. 1).
      • Haick H.
      • Broza Y.Y.
      • Mochalski P.
      • Ruzsanyi V.
      • Amann A.
      Assessment, origin, and implementation of breath volatile cancer markers.
      In this setting, free radicals and reactive oxygen species (ROS), characterized by an unpaired electron in the outer shell originating from endogenous as well as exogenous sources (i.e., cigarette smoke), can interact with protein and fatty acids to produce VOCs (Fig. 1).
      • Haick H.
      • Broza Y.Y.
      • Mochalski P.
      • Ruzsanyi V.
      • Amann A.
      Assessment, origin, and implementation of breath volatile cancer markers.
      Currently, more than 3000 VOCs have been detected in the human breath.
      • Haick H.
      • Broza Y.Y.
      • Mochalski P.
      • Ruzsanyi V.
      • Amann A.
      Assessment, origin, and implementation of breath volatile cancer markers.
      One distinctive feature of VOCs is that they are individually detected in minimal concentrations by sophisticated analytical equipments.
      • D'Amico A.
      • Pennazza G.
      • Santonico M.
      • et al.
      An investigation on electronic nose diagnosis of lung cancer.
      Another characteristic of the VOCs is that they are exhaled in human breath according to an equilibrium concentration resulting from their “fat-to-blood’’ and ‘‘blood-to-air’’ partition coefficients specific to the compartments where these products of cellular metabolism can be interchanged.
      • Haick H.
      • Broza Y.Y.
      • Mochalski P.
      • Ruzsanyi V.
      • Amann A.
      Assessment, origin, and implementation of breath volatile cancer markers.
      Table 1Lung Cancer–Related VOCs According to Recent Literature
      CompoundsPossible Endogenous SourceMain Products and/or DerivativesExogenous Origin
      Alkanes alkenesOxidative stress (PUFA peroxidation)Ethane

      Pentane

      Heptane

      Octane

      Decane

      Undecane

      Dodecane

      Nonadecane

      Isoprene

      2,2,4-Trimethylexane

      Propane

      Eicosane
      Natural, plastics or petrol/fuels
      AlcoholsHydrocarbon metabolism

      Absorbed through GI tract
      Propanol

      Butanol

      2-Ethyl-1-hexanol

      4-Penten-2-ol

      Ethanol

      Methanol

      Heptadecanol
      Natural, diet or disinfectants
      AldehydesMetabolism of alcohols

      Lipid peroxidation
      Propanal

      Butanal

      Pentanal

      Hexanal

      Heptanal

      Octanal

      Nonanal

      Formaldehyde

      Acetaldehyde
      Natural, diet or waste products Smoking
      KetonesFatty acid oxidation

      Protein metabolism
      Acetone

      Butanone

      Pentanone

      Hexanone

      Heptanone

      Benzophenone

      Hendecanone

      Pentadecanone

      Heptadecanone
      Natural, diet, waste products or drugs/fragrances/paint
      Carboxylic acidsMetabolism of amino acidsBenzoic acid

      Propanoic acid

      Acetic acid
      Food preservatives, solvents, polymers
      EstersMetabolic pathway of alcohols and acidsEthanoate

      Propanoate

      Acetate
      Fatty oils, natural wax, fruit essential oils
      NitrilesAcetonitrile

      Azulencarbonitrile
      Smoking
      Aromatic compoundsBenzene

      Toluene

      Styrene

      2,5 Dimethylfuran

      Anthracene

      Dimethylnaphtalene
      Petrol, smoking, natural (styrene),tar, oil
      TerpensLimonene

      Camphor
      Natural or cosmetics
      From Haick et al.
      • Haick H.
      • Broza Y.Y.
      • Mochalski P.
      • Ruzsanyi V.
      • Amann A.
      Assessment, origin, and implementation of breath volatile cancer markers.
      PUFA, polyunsaturated fatty acids; GI, gastrointestinal.
      Figure thumbnail gr1
      Figure 1Metabolic pathways leading to the generation of volatile organic compounds (VOCs. Oxidative stress through the activation of cytochrome p450 determines the specific pattern of breath VOCs (see text). Reproduced from Haick et al.
      • Haick H.
      • Broza Y.Y.
      • Mochalski P.
      • Ruzsanyi V.
      • Amann A.
      Assessment, origin, and implementation of breath volatile cancer markers.
      with permission. ROS, reactive oxygen species; PUFA, polyunsaturated fatty acid; VOC, volatile organic compound.
      Accordingly, VOC chemical and physical properties, as well as many physiologic and pathologic conditions, may potentially interfere with the concentration of these compounds both in the alveoli and in the proximal airway.
      • Haick H.
      • Broza Y.Y.
      • Mochalski P.
      • Ruzsanyi V.
      • Amann A.
      Assessment, origin, and implementation of breath volatile cancer markers.
      As an example, the specific VOC lipophilic status can affect its diffusion through cell membranes; in fact, less lipophilic VOCs are exchanged in the airway thereby emphasizing the role of the airway in the gas exchange process.
      • Haick H.
      • Broza Y.Y.
      • Mochalski P.
      • Ruzsanyi V.
      • Amann A.
      Assessment, origin, and implementation of breath volatile cancer markers.
      In addition, the quantity of VOCs in the alveoli depends from their retention in the lung, in turn directly proportional to the alveolar clearance.
      • Haick H.
      • Broza Y.Y.
      • Mochalski P.
      • Ruzsanyi V.
      • Amann A.
      Assessment, origin, and implementation of breath volatile cancer markers.

      The Canine Model of Breath Analysis

      Canine scent detection has been investigated for several decades due to its potential to identify breath complex chemical compounds present in parts per trillion.
      • McCulloch M.
      • Jezierski T.
      • Broffman M.
      • Hubbard A.
      • Turner K.
      • Janecki T.
      Diagnostic accuracy of canine scent detection in early- and late-stage lung and breast cancers.
      In 2006, McCulloch et al.
      • McCulloch M.
      • Jezierski T.
      • Broffman M.
      • Hubbard A.
      • Turner K.
      • Janecki T.
      Diagnostic accuracy of canine scent detection in early- and late-stage lung and breast cancers.
      published a trial determining the accuracy of the canine olfactory system of trained household dogs by comparing breath samples from lung and breast cancer patients to healthy individuals.
      • McCulloch M.
      • Jezierski T.
      • Broffman M.
      • Hubbard A.
      • Turner K.
      • Janecki T.
      Diagnostic accuracy of canine scent detection in early- and late-stage lung and breast cancers.
      Sensitivity and specificity of the canine olfactory system compared to conventional diagnostic pathways were 99% (95% confidence interval: 99–100) and 99% (95% confidence interval: 96–100), respectively. To curb the enthusiasm for this intriguing modality for early lung cancer detection, two major limitations arguing against routinely using sniffer dogs emerged from subsequent contributions. First, the uncertainty as to which compounds the canine olfactory system may be stimulated by and, second, the unreliable collaborative attitude expressed by the animal due to motivation or other confounding factors.
      • Jezierski T.
      • Walczak M.
      • Ligor T.
      • Rudnicka J.
      • Buszewski B.
      Study of the art: canine olfaction used for cancer detection on the basis of breath odour. Perspectives and limitations.
      However, the ability of trained dogs to detect lung cancer–related scent patterns represented a novel concept which links canine scent detection to pattern recognition–based models of the currently available e-nose devices.
      • Boedeker E.
      • Friedel G.
      • Walles T.
      Sniffer dogs as part of a bimodal bionic research approach to develop a lung cancer screening.
      In addition, the use of canine olfactory receptors as a biophysical template to structure e-noses is being investigated (see below).
      • Pomerantz A.
      • Blachman-Braun R.
      • Galnares-Olalde J.A.
      • Berebichez-Fridman R.
      • Capurso-García M.
      The possibility of inventing new technologies in the detection of cancer by applying elements of the canine olfactory apparatus.

      A Bioengineered Sensorial Platform

      The exhaled breath can be analyzed based on two distinct albeit complementary approaches (Fig. 2)
      • van der Schee M.P.
      • Paff T.
      • Brinkman P.
      • van Aalderen W.M.C.
      • Haarman E.G.
      • Sterk P.J.
      Breathomics in lung disease.
      : (1) VOC identification, and (2) VOC patterning. The first approach is aimed at identifying characteristic biomarkers associated with specific diseases through selective sensors for VOCs.
      • Haick H.
      • Broza Y.Y.
      • Mochalski P.
      • Ruzsanyi V.
      • Amann A.
      Assessment, origin, and implementation of breath volatile cancer markers.
      The instrumentation used to identify individual VOCs is based on the mainstream of analytical chemistry, mainly GC coupled with mass spectrometry (GC-MS).
      • van der Schee M.P.
      • Paff T.
      • Brinkman P.
      • van Aalderen W.M.C.
      • Haarman E.G.
      • Sterk P.J.
      Breathomics in lung disease.
      Conversely, the second approach looks for exhaled breath fingerprint through cross-reactive sensors, aiming at defining specific patterns of disease-related VOCs — without proceeding to their individual identification.
      • Haick H.
      • Broza Y.Y.
      • Mochalski P.
      • Ruzsanyi V.
      • Amann A.
      Assessment, origin, and implementation of breath volatile cancer markers.
      Figure thumbnail gr2
      Figure 2The two complementary methods for breath analysis (see text). The individual VOCs are identified through GC-MS according to their physical features (i.e., mass to charge ratio) which are then compared to a reference library (top). Alternatively, the characteristic patterns of VOCs are recognized through the e-nose technology and linked to specific disease conditions (bottom). Reproduced from van der Schee et a.
      • van der Schee M.P.
      • Paff T.
      • Brinkman P.
      • van Aalderen W.M.C.
      • Haarman E.G.
      • Sterk P.J.
      Breathomics in lung disease.
      with permission. VOC, volatile organic compound; GC, gas chromatography; MS, mass spectrometry.

      VOC Collection and Sampling

      VOCs can either be collected by “trapping” (by means of sorbent materials, within a chamber, or dehumidifier) or can be directly delivered to a measurement device (i.e., sensor) (Fig. 3).
      • Haick H.
      • Broza Y.Y.
      • Mochalski P.
      • Ruzsanyi V.
      • Amann A.
      Assessment, origin, and implementation of breath volatile cancer markers.
      VOC collection before delivery exposes several drawbacks ranging from the loss of VOCs to potential contamination of the gaseous sample.
      • Haick H.
      • Broza Y.Y.
      • Mochalski P.
      • Ruzsanyi V.
      • Amann A.
      Assessment, origin, and implementation of breath volatile cancer markers.
      Conversely, direct delivery to the measurement unit allows for interaction with the sensor array by inducing either an electrical or optical changes then bound to be transduced into signals.
      • Haick H.
      • Broza Y.Y.
      • Mochalski P.
      • Ruzsanyi V.
      • Amann A.
      Assessment, origin, and implementation of breath volatile cancer markers.
      The interaction between VOCs and sensors will be specific or semiselective depending on the need for VOC identification (i.e., selective or lock-and-key sensors) as opposed to patterning (i.e., cross-reactive sensors).
      • Haick H.
      • Broza Y.Y.
      • Mochalski P.
      • Ruzsanyi V.
      • Amann A.
      Assessment, origin, and implementation of breath volatile cancer markers.
      Figure thumbnail gr3
      Figure 3The stepwise process leading to breath analysis. The modalities of breath sample collection, preparation, measurement and evaluation through predictive models are illustrated (see text). Reproduced from Haick et al.
      • Haick H.
      • Broza Y.Y.
      • Mochalski P.
      • Ruzsanyi V.
      • Amann A.
      Assessment, origin, and implementation of breath volatile cancer markers.
      with permission. VOC, volatile organic compound.

      VOC Analysis and Identification

      To analyze VOCs in human breath, the ideal sensors should capture minimal absolute VOC concentrations and variations thereof, rapidly return to baseline status if not exposed to VOCs, and be so easily manufactured and low-priced to be available in large disposable quantities.
      • Haick H.
      • Broza Y.Y.
      • Mochalski P.
      • Ruzsanyi V.
      • Amann A.
      Assessment, origin, and implementation of breath volatile cancer markers.
      For VOC identification, the gas sample is transferred into GC-MS where it is analyzed according to the time to elution (i.e., absorption) in the chromatography column and to the mass-to-charge ratio.
      • van der Schee M.P.
      • Paff T.
      • Brinkman P.
      • van Aalderen W.M.C.
      • Haarman E.G.
      • Sterk P.J.
      Breathomics in lung disease.
      The quantification of VOCs by GC-MS is an expensive, time-consuming process which demands expert personnel as well as dedicated laboratory facilities.
      • Haick H.
      • Broza Y.Y.
      • Mochalski P.
      • Ruzsanyi V.
      • Amann A.
      Assessment, origin, and implementation of breath volatile cancer markers.
      • van der Schee M.P.
      • Paff T.
      • Brinkman P.
      • van Aalderen W.M.C.
      • Haarman E.G.
      • Sterk P.J.
      Breathomics in lung disease.

      VOC Patterning: Sensor Features

      From a technical standpoint, the starting point for capturing a signature of the whole exhaled breath consists of the use of a large number of nonselective sensors combined in the so-called sensor arrays.
      • Phillips M.
      Method for the collection and assay of volatile organic compounds in breath.

      ReCIVA Breath Sampler. https://www.owlstonemedical.com/products/reciva/. Accessed January 18, 2018.

      • Nakhleh M.K.
      • Amal H.
      • Jeries R.
      • et al.
      Diagnosis and classification of 17 diseases from 1404 subjects via pattern analysis of exhaled molecules.
      Multiple sensor arrays produce a multidimensional output which is then analyzed with pattern recognition techniques specific to multivariate data analysis. Based on the available gas sensor arrays, dubbed e-noses used by different research groups and enterprises all over the world can be distinguished into the e-nose and the hybrid e-nose. The e-nose group includes the sensor arrays based on conductive materials (i.e., thin oxide, gold nanoparticles, polymers) and electroacoustic sensors (i.e., Quartz Microbalance [QMB] or Surface Acoustic Wave Sensors).
      • Haick H.
      • Broza Y.Y.
      • Mochalski P.
      • Ruzsanyi V.
      • Amann A.
      Assessment, origin, and implementation of breath volatile cancer markers.
      • Peng G.
      • Hakim M.
      • Broza Y.Y.
      • et al.
      Detection of lung, breast, colorectal, and prostate cancers from exhaled breath using a single array of nanosensors.
      • Machado R.F.
      • Laskowski D.
      • Deffenderfer O.
      • et al.
      Detection of lung cancer by sensor array analyses of exhaled breath.
      • Montuschi P.
      • Mores N.
      • Trové A.
      • Mondino C.
      • Barnes P.J.
      The electronic nose in respiratory medicine.
      • Hakim M.
      • Billan S.
      • Tisch U.
      • et al.
      Diagnosis of head-and-neck cancer from exhaled breath.
      • Blatt R.
      • Bonarini A.
      • Calabro E.
      • Della Torre M.
      • Matteucci M.
      • Pastorino U.
      Lung cancer identification by an electronic nose based on an array of MOS sensors.
      • Zetola N.M.
      • Modongo C.
      • Matsiri O.
      • et al.
      Diagnosis of pulmonary tuberculosis and assessment of treatment response through analyses of volatile compound patterns in exhaled breath samples.
      • Santonico M.
      • Pennazza G.
      • Grasso S.
      • D'Amico A.
      • Bizzarri M.
      Design and test of a biosensor-based multisensorial system: a proof of concept study.
      The colorimetric sensor arrays represent another category of sensors characterized by exposing the VOCs to a series of chemoresponsive dyes, thereby generating color changes visible by the naked eye or at spectroscopy.
      • Mazzone P.J.
      • Wang X.F.
      • Xu Y.
      • et al.
      Exhaled breath analysis with a colorimetric sensor array for the identification and characterization of lung cancer.
      • Mazzone P.J.
      • Wang X.F.
      • Lim S.
      • et al.
      Progress in the development of volatile exhaled breath signatures of lung cancer.
      The other category of breath analyzers could be called “hybrid e-noses” because these devices are based on the integration of sensor performing according to the traditional methods of the analytical chemistry (i.e., spectroscopic approach) and a fingerprinting approach.
      • Saidi T.
      • Moufid M.
      • Zaim O.
      • EL Bari N.
      • Ionescu R.
      • Bouchikhi B.
      artificial sensory systems combined with UV-Vis spectrophotometry as a robust approach for VOCs analysis of human urine and exhaled breath.
      To summarize, the best standards for breathprinting are obtained by the following measure chain: (1) a device able to collect exhaled breath onto a cartridge which can be transported and stored; (2) an instrument for desorbing the exhaled breath into the sensor cell; and (3) a versatile sensor system including several nonselective sensing elements able to measure biological exhalates. Currently, two systems seem to abide by the above requirements. The first system is manufactured by Owlstone Inc., who has created a device for exhaled breath sampling known as ReCIVA as well as the Lonestar breath analyser.
      • Horvath I.
      • Lazar Z.
      • Gyulai N.
      • Kollai M.
      • Losonczy G.
      Exhaled biomarkers in lung cancer.

      ReCIVA Breath Sampler. https://www.owlstonemedical.com/products/reciva/. Accessed January 18, 2018.

      LONESTAR (2017) Lonestar Gas Analyzer. http://www.owlstonenanotech.com/lonestar (2017). Accessed January 30, 2017.

      The latter is a commercially available device based on a hybrid e-nose technology. The second system has been manufactured by a group from the University Campus Bio-Medico of Rome, Italy. It is based on a measure chain including a device for exhaled breath collection on a cartridge known as the Pneumopipe (European patent n. 12425057.2)
      • Pennazza G.
      • et al.
      Measure chain for exhaled breath collection and analysis: a novel approach suitable for frail respiratory patients.
      ; a desorbing unit able to thermally desorb the cartridge content into the sensor cell (known as the Breathstiller); and a gas and liquid sensor array able to analyze the volatile and liquid part of any biological fluid, known as BIONOTE.
      • Santonico M.
      • Pennazza G.
      • Grasso S.
      • D'Amico A.
      • Bizzarri M.
      Design and test of a biosensor-based multisensorial system: a proof of concept study.
      The BIONOTE (BIOsensor-based multisensorial system for mimicking Nose, Tongue and Eyes) comprises in a single device a multisensorial platform aimed at weighing VOCs by using an array of Qujarz microbalance sensors using anthocyanins as chemical interactive material. BIONOTE can also perform the electrochemical or the optical analysis of the liquid part of the patient’ sample.
      • Santonico M.
      • Pennazza G.
      • Grasso S.
      • D'Amico A.
      • Bizzarri M.
      Design and test of a biosensor-based multisensorial system: a proof of concept study.
      The potential use of such multisensorial platform is reported later in this article.

      A Bioengineered Sensorial Platform: The Breathprint

      Fingerprints can be univocally associated to a single individual for his/her identification. This is a well-known technique based on three fundamental elements: a standard list of features to be used as reference for each comparison, a huge library of individual fingerprints, and an effective method for fingerprinting. Similarly, exhaled breath fingerprint (also known as breathprint) should define the individual health-state, as preconized by several studies in the last twenty years.
      • Fens N.
      • Zwinderman A.H.
      • van der Schee M.P.
      • et al.
      Exhaled breath profiling enables discrimination of chronic obstructive pulmonary disease and asthma.
      • Buszewski B.
      • Kesy M.
      • Ligor T.
      • Amann A.
      Human exhaled air analytics: biomarkers of diseases.
      • Friedrich M.J.
      Scientists seek to sniff out diseases: electronic “noses” may someday be diagnostic tools.
      • Kharitonov S.A.
      • Barnes P.J.
      Exhaled markers of pulmonary disease.
      • D’Amico A.
      • Di Natale C.
      • Paolesse R.
      • et al.
      Olfactory systems for medical applications.
      • Peng G.
      • Tisch U.
      • Adams O.
      • et al.
      Diagnosing lung cancer in exhaled breath using gold nanoparticles.
      The breathprint is obtained in the presence of a device for breathprint capturing (collection and measurement), a method for its analysis and a breathprint library.
      • Beauchamp J.D.
      • Pleil J.D.
      Simply breath-taking? Developing a strategy for consistent breath sampling.
      • Pennazza G.
      • Santonico M.
      • Finazzi Agrò A.
      Narrowing the gap between breathprinting and disease diagnosis, a sensor perspective.
      • Horváth I.
      • Barnes P.J.
      • Loukides S.
      • et al.
      A European Respiratory Society technical standard: exhaled biomarkers in lung disease.
      As a consequence, the refinement in the implementation of these three principals have marked the evolution of breathprinting technology. As an example, the methods used for exhaled breath collection have accounted for the influence of the environment background, the selection of the exhaled breath volume to be sampled, and the duration and modality of sampling (i.e., blowing as opposed to normal breathing).
      • Horváth I.
      • Barnes P.J.
      • Loukides S.
      • et al.
      A European Respiratory Society technical standard: exhaled biomarkers in lung disease.
      Also, some confounding and/or influential factors, such as smoking, food intake, and other habits, have been considered to evaluate and possibly limit their effects on the collected sample.
      • Horváth I.
      • Barnes P.J.
      • Loukides S.
      • et al.
      A European Respiratory Society technical standard: exhaled biomarkers in lung disease.
      Exhaled breath collection can be performed with a direct delivery of the exhalate from the subject into the device measurement cell, or with an indirect modality by using sampling media.
      • Pennazza G.
      • et al.
      Measure chain for exhaled breath collection and analysis: a novel approach suitable for frail respiratory patients.
      The latter technique is the most frequently used, whereas sampling bags or adsorbing cartridges are the two most frequently used media.
      • Horváth I.
      • Barnes P.J.
      • Loukides S.
      • et al.
      A European Respiratory Society technical standard: exhaled biomarkers in lung disease.
      Obviously, the selection of the sampling medium plays an important role with respect to its subsequent analysis as it contributes to amplifying, diluting, or modifying the sample itself. From a patient point of view, breathing into a device which autonomously conveys the sample in a cartridge is less invasive than breathing into a bag. Moreover, from an experimental point of view, the possibility of storing and transporting exhaled breath samples in a cartridge instead of a fragile (in terms of variability) bag represents an added value. Finally, from a technical point of view, the use of a preconcentration technique (i.e., through an adsorbing cartridge) improves the sensor sensitivity offering a better resolution at low concentration values. In addition, an ad hoc device aimed at collecting the exhaled breath into a cartridge must be manufactured. Currently, three such devices are available for clinical use, one made by the University Campus Bio-Medico of Rome, Italy, one by the Menssana research team, and one from Owlstone Inc.
      • Phillips M.
      Method for the collection and assay of volatile organic compounds in breath.

      ReCIVA Breath Sampler. https://www.owlstonemedical.com/products/reciva/. Accessed January 18, 2018.

      • Pennazza G.
      • et al.
      Measure chain for exhaled breath collection and analysis: a novel approach suitable for frail respiratory patients.

      Phillips M. System and method for remote collection and analysis of volatile organic components in breath. US patent 20130253358A1. 26 September 2013.

      Results of Breathprinting in Non-Neoplastic Conditions

      E-nose technology has been proven to have important discriminatory and classificatory properties in several mainly respiratory chronic diseases. A breathprint easily distinguishes asthma from chronic obstructive pulmonary disease patients and from controls.
      • Fens N.
      • Zwinderman A.H.
      • van der Schee M.P.
      • et al.
      Exhaled breath profiling enables discrimination of chronic obstructive pulmonary disease and asthma.
      • Montuschi P.
      • Santonico M.
      • Mondino C.
      • et al.
      Diagnostic performance of an electronic nose, fractional exhaled nitric oxide, and lung function testing in asthma.
      • de Vries R.
      • Brinkman P.
      • van der Schee M.P.
      • et al.
      Integration of electronic nose technology with spirometry: validation of a new approach for exhaled breath analysis.
      Besides differing from that of controls, the breathprint also distinguishes cirrhotic from non-cirrhotic patients and significantly changes in patients with decompensated liver cirrhosis.
      • De Vincentis A.
      • Pennazza G.
      • Santonico M.
      • et al.
      Breath-print analysis by e-nose for classifying and monitoring chronic liver disease: a proof-of-concept study.
      • De Vincentis A.
      • Pennazza G.
      • Santonico M.
      • et al.
      Breath-print analysis by e-nose may refine risk stratification for adverse outcomes in cirrhotic patients.
      At a 2-year follow-up examination, two breathprint patterns corresponding to the largest and the smallest amounts of produced VOCs were associated with the worst survival, adding to the prognostic capacity of traditional indexes such as the Child Plug category.
      • De Vincentis A.
      • Pennazza G.
      • Santonico M.
      • et al.
      Breath-print analysis by e-nose may refine risk stratification for adverse outcomes in cirrhotic patients.
      E-nose technology has been successfully used to distinguish patients with several neurologic and mental disorders from controls.
      • D’Amico A.
      • Di Natale C.
      • Paolesse R.
      • et al.
      Olfactory systems for medical applications.
      • Dragonieri S.
      • Quaranta V.N.
      • Carratu P.
      • et al.
      An electronic nose may sniff out amyotrophic lateral sclerosis.
      • Mazzatenta A.
      • Pokorski M.
      • Sartucci F.
      • Domenici L.
      • Di Giulio C.
      Volatile organic compounds (VOCs) fingerprint of Alzheimer’s disease.
      • Tisch U.
      • Schlesinger I.
      • Ionescu R.
      • et al.
      Detection of Alzheimer’s and Parkinson’s disease from exhaled breath using nanomaterial-based sensors.
      • Bach J.-P.
      • Gold M.
      • Mengel D.
      • et al.
      Measuring compounds in exhaled air to detect Alzheimer’s Disease and Parkinson’s Disease.
      Experience from patients with non-malignant conditions has emphasized the potentially significant impact of age, gender, and comorbidities on the reproducibility of e-nose measurements to facilitate an unconfounded estimation of cancer-related breathprint.
      • Antonelli Incalzi R.
      • Pennazza G.
      • Scarlata S.
      • et al.
      Comorbidity modulates non invasive ventilation-induced changes in breath print of obstructive sleep apnea syndrome patients.
      • Dragonieri S.
      • Quaranta V.N.
      • Carratu P.
      • Ranieri T.
      • Resta O.
      Influence of age and gender on the profile of exhaled volatile organic compounds analyzed by an electronic nose.
      In this setting, e-nose technology might also surrogate spirometry in people who are unable to comply with the rigorous procedural standards of spirometry. In fact, in approximately one of four people older than 65 years, spirometry cannot be completed mainly due to illiteracy, sarcopenia, severe bronchial obstruction, or cognitive impairment.
      • Bellia V.
      • Pistelli R.
      • Catalano F.
      • et al.
      Quality control of spirometry in the elderly. The SA.R.A. study. SAlute Respiration nell’Anziano = Respiratory Health in the Elderly.

      Results of Breathprinting for Early Diagnosis of Lung Cancer

      In an effort to achieve an early diagnosis of lung cancer, many studies were aimed at validating noninvasive diagnostic methods to find an effective test for screening high-risk populations (Table 2). In 2011, investigators of the National Lung Screening Trial reported a 20% reduction in lung cancer–specific mortality by using low-dose computed tomography (LDCT) to screen populations at high risk for developing lung cancer.
      • Aberle D.R.
      • Adams A.M.
      • Berg C.D.
      • et al.
      Reduced lung-cancer mortality with low-dose computed tomographic screening.
      Radiation exposure, the occurrence of false-positive results leading to unnecessary invasive diagnostic procedures and the reported over-diagnosis of slow-growing tumors, along with the paradoxical encouragement of smoking represent potential arguments against the implementation of LDCT screening.
      • Krantz S.B.
      • Meyers B.F.
      Health risks from computed tomographic screening.
      • Crucitti P.
      • Gallo I.F.
      • Santoro G.
      • Mangiameli G.
      Lung cancer screening with low dose CT: experience at Campus Bio-Medico of Rome on 1500 patients.
      • Mangiameli G.
      • Longo F.
      • Grasso R.F.
      • Iacopino A.
      • Rocco R.
      • Crucitti P.
      Focus on lung cancer screening program at Campus Bio-Medico of Rome: update on more than 3250 patients.
      Strategies for reducing false positives resulting from LDCT screening consist of the periodic revision of the guidelines for the interpretation and management of nodules (i.e., volumetric assessment of the nodule) and the research of possible complementary biomarkers.
      • Devaraj A.
      • van Ginneken B.
      • Nair A.
      • Baldwin D.
      Use of volumetry for lung nodule management: theory and practice.
      • Sozzi G.
      • Boeri M.
      • Rossi M.
      • et al.
      Clinical utility of a plasma-based miRNA signature classifier within computed tomography lung cancer screening: a correlative MILD trial study.
      In this setting, many studies tried to validate the efficacy of the assessment of VOCs in the exhaled breath as a diagnostic tool for lung cancer. In 2012, Ehmann et al.
      • Ehmann R.
      • Boedeker E.
      • Friedrich U.
      • et al.
      Canine scent detection in the diagnosis of lung cancer: revisiting a puzzling phenomenon.
      published an interesting paper in which they analyzed the capability of four trained family dogs in detecting lung cancer. Their study was a prospective, blinded, clinical trial in which dogs produced 125 breath samples yielding an overall sensitivity of 71% and a specificity of 93%, thus confirming the existence of a scent pattern associated with lung cancer.
      • Ehmann R.
      • Boedeker E.
      • Friedrich U.
      • et al.
      Canine scent detection in the diagnosis of lung cancer: revisiting a puzzling phenomenon.
      Expectedly, one major limitation of this study was the low reproducibility of the test. In the past decades, several artificial olfaction systems (i.e., e-nose technology) based on nonselective chemical sensors aimed at measuring and comparing VOC patterns (breathprint) have been proposed in the literature to obtain a precise identification of the compounds observed in exhaled breath of lung cancer patients.
      • Pennazza G.
      • Santonico M.
      • Finazzi Agrò A.
      Narrowing the gap between breathprinting and disease diagnosis, a sensor perspective.
      • Li W.
      • Liu H.Y.
      • Jia Z.R.
      • et al.
      Advances in the early detection of lung cancer using analysis of volatile organic compounds: from imaging to sensors.
      Such technologies differ from each other in many respects, from the working principle to the sensing material and the array composition. In 2015, McWilliams et al.
      • McWilliams A.
      • Beigi P.
      • Srinidhi A.
      • Lam S.
      • MacAulay C.E.
      sex and smoking status effects on the early detection of early lung cancer in high-risk smokers using an electronic nose.
      collected and analyzed breath samples both from lung cancer patients and high-risk smokers without cancer. The investigators found that with the e-nose device known as the Cyranose 320 they could distinguish lung cancer patients from high-risk control subjects with a better than 80% accuracy, thereby concluding that e-nose technology could be used as a noninvasive tool for screening individuals at increased risk for lung cancer.
      • McWilliams A.
      • Beigi P.
      • Srinidhi A.
      • Lam S.
      • MacAulay C.E.
      sex and smoking status effects on the early detection of early lung cancer in high-risk smokers using an electronic nose.
      Later, Rocco et al.
      • Rocco R.
      • Antonelli Incalzi R.
      • Pennazza G.
      • et al.
      BIONOTE e-nose technology may reduce false positives in lung cancer screening programmes.
      validated an artificial olfactory system called BIONOTE to detect lung cancer in patients who were enrolled in a lung cancer screening program. The investigators examined 100 individuals who were considered high-risk for lung cancer with the BIONOTE yielding an overall sensitivity and specificity of 86% and 95%, respectively, thereby delineating a substantial difference between breathprints from lung cancer patients as opposed to healthy individuals.
      • Rocco R.
      • Antonelli Incalzi R.
      • Pennazza G.
      • et al.
      BIONOTE e-nose technology may reduce false positives in lung cancer screening programmes.
      Table 2Sensitivity and Specificity Outcomes From Different Models of Breath Analysis
      Method of Breath AnalysisSensitivity (%)Specificity (%)AccuracyAccuracy
      E-nose based on colorimetric sensor array technology; C-statistic for the distinction between adenocarcinoma and squamous cell = 0.864.26,67,82
      Adca
      Accuracy
      E-nose based on colorimetric sensor array technology; C-statistic for the distinction between adenocarcinoma and squamous cell = 0.864.26,67,82


      Squam
      Accuracy
      E-nose based on colorimetric sensor array technology; C-statistic for the distinction between adenocarcinoma and squamous cell = 0.864.26,67,82


      Stage I
      Canine71–9982–99
      GC-MS51–9067–10069–97888888
      E-nose70–9373–10080–100838685
      E-nose + GC-MS10080–9688–94
      Adca, adenocarcinoma; Squam, squamous cell carcinoma; GC-MS, gas chromatography–mass spectrometry.
      a E-nose based on colorimetric sensor array technology; C-statistic for the distinction between adenocarcinoma and squamous cell = 0.864.
      • Mazzone P.J.
      • Wang X.F.
      • Xu Y.
      • et al.
      Exhaled breath analysis with a colorimetric sensor array for the identification and characterization of lung cancer.
      • Peled N.
      • Hakim M.
      • Bunn Jr., P.A.
      • et al.
      Non-invasive breath analysis of pulmonary nodules.
      • Nardi-Agmon I.
      • Peled N.
      Exhaled breath analysis for the early detection of lung cancer: recent developments and future prospects.
      In 2012, Barash et al.
      • Barash O.
      • Peled N.
      • Tisch U.
      • Bunn Jr., P.A.
      • Hirsch F.R.
      • Haick H.
      Classification of lung cancer histology by gold nanoparticle sensors.
      used gold nanoparticle sensors to distinguish VOCs from cell lines of healthy individuals compared to different lung cancer histotypes. The investigators succeeded in discriminating between lung carcinoma and healthy cells, between NCLC and SCLC cells, and between adenocarcinoma and squamous cell carcinoma with an accuracy ranging between 9% and 96%.
      • Barash O.
      • Peled N.
      • Tisch U.
      • Bunn Jr., P.A.
      • Hirsch F.R.
      • Haick H.
      Classification of lung cancer histology by gold nanoparticle sensors.
      In addition, the possibility to identify genetic mutations to guide the oncologists’ practice was shown by a group of investigators who identified the breathprints associated with EGFR and KRAS mutations as well as with anaplastic lymphoma kinase and p53 rearrangements.
      • Peled N.
      • Barash O.
      • Tisch U.
      • Ionescu R.
      • Broza Y.Y.
      Volatile fingerprints of cancer specific genetic mutations.
      • Davies M.P.A.
      • Barash O.
      • Jeries R.
      • et al.
      Unique volatolomic signatures of TP53 and KRAS in lung cells.
      Recently, Shlomi et al.
      • Shlomi D.
      • Abud M.
      • Liran O.
      • Bar J.
      • Gai-Mor N.
      • Ilouze M.
      • et al.
      Detection of lung cancer and EGFR mutation by electronic nose system.
      reported that patients with early lung cancer could be discriminated from patients with benign pulmonary nodules with a sensitivity, specificity, and accuracy of 75%, 93%, and 87%, respectively. Both positive predictive values and negative predictive values were high (approximately 88%), indicating the efficiency and suitability of the test despite the small number of patients enrolled in the study.
      • Shlomi D.
      • Abud M.
      • Liran O.
      • Bar J.
      • Gai-Mor N.
      • Ilouze M.
      • et al.
      Detection of lung cancer and EGFR mutation by electronic nose system.
      Today, the scientific interest in e-nose technology is growing strongly even in the absence of absolute scientific evidence that could drive clinical practice worldwide. Future studies will need to focus on the validating e-nose technology to replicate the same high percentages of specificity and sensitivity reported in the relatively low numbered series. E-nose technology must also be made applicable on a large scale, meaning devices must be user friendly, less expensive, and capable to rapidly yield a “yes/no” result.
      Given the available knowledge, several potential applications of e-nose technology could be anticipated. These include the verification of smoking cessation in patients enrolled in lung cancer screening programs along with the ability to identify high-risk individuals for LDCT.
      • Rocco R.
      • Antonelli Incalzi R.
      • Pennazza G.
      • et al.
      BIONOTE e-nose technology may reduce false positives in lung cancer screening programmes.
      • Schumer E.M.
      • Trivedi J.R.
      • van Berkel V.
      • Black M.C.
      • Li M.
      • Fu X.A.
      • et al.
      High sensitivity for lung cancer detection using analysis of exhaled carbonyl compounds.
      • Incalzi R.A.
      • Pennazza G.
      • Scarlata S.
      • Santonico M.
      • Petriaggi M.
      • Chiurco D.
      • et al.
      Reproducibility and respiratory function correlates of exhaled breath fingerprint in chronic obstructive pulmonary disease.
      In addition, the e-nose could be used to confirm a suspected pulmonary nodule before scheduling an invasive surgical procedure.
      • Shlomi D.
      • Abud M.
      • Liran O.
      • Bar J.
      • Gai-Mor N.
      • Ilouze M.
      • et al.
      Detection of lung cancer and EGFR mutation by electronic nose system.
      • Santonico M.
      • Pennazza G.
      • Grasso S.
      • D'Amico A.
      • Bizzarri M.
      Design and test of a biosensor-based multisensorial system: a proof of concept study.
      • Pennazza G.
      • Santonico M.
      • Finazzi Agrò A.
      Narrowing the gap between breathprinting and disease diagnosis, a sensor perspective.
      • Peled N.
      • Hakim M.
      • Bunn Jr., P.A.
      • et al.
      Non-invasive breath analysis of pulmonary nodules.
      After surgery, breath analysis may serve to determine if and when to perform a follow-up computed tomographic scan or positron-emission tomography.
      • Rocco R.
      • Antonelli Incalzi R.
      • Pennazza G.
      • et al.
      BIONOTE e-nose technology may reduce false positives in lung cancer screening programmes.
      • Schumer E.M.
      • Black M.C.
      • Bousamra 2nd, M.
      • Trivedi J.R.
      • Li M.
      • Fu X.A.
      • et al.
      Normalization of exhaled carbonyl compounds after lung cancer resection.
      When surgery is not indicated and a diagnostic biopsy of a suspected lesion would be hazardous, the e-nose can support the clinical algorithms in establishing malignancy before administering alternative treatments, such as stereotactic body radiation therapy.
      • Shlomi D.
      • Abud M.
      • Liran O.
      • Bar J.
      • Gai-Mor N.
      • Ilouze M.
      • et al.
      Detection of lung cancer and EGFR mutation by electronic nose system.
      • Rocco R.
      • Antonelli Incalzi R.
      • Pennazza G.
      • et al.
      BIONOTE e-nose technology may reduce false positives in lung cancer screening programmes.
      • Barash O.
      • Peled N.
      • Tisch U.
      • Bunn Jr., P.A.
      • Hirsch F.R.
      • Haick H.
      Classification of lung cancer histology by gold nanoparticle sensors.
      Another interesting perspective is represented by the use of the e-nose to identify lung cancer–related genetic mutations and to support Response Evaluation Criteria in Solid Tumors criteria to assess the response to medical treatments.
      • Shlomi D.
      • Abud M.
      • Liran O.
      • Bar J.
      • Gai-Mor N.
      • Ilouze M.
      • et al.
      Detection of lung cancer and EGFR mutation by electronic nose system.
      • Broza Y.Y.
      • Kremer R.
      • Tisch U.
      • Gevorkyan A.
      • Shiban A.
      • Best L.A.
      • et al.
      A nanomaterial-based breath test for short-term follow-up after lung tumor resection.
      • Nardi-Agmon I.
      • Abud-Hawa M.
      • Liran O.
      • et al.
      Exhaled breath analysis for monitoring response to treatment in advanced lung cancer.
      However, the low number used in the series exploring the value of e-nose technology and the lack of standardized and miniaturized devices enabling physicians to complete the sample collection and data analysis in real time appear to be major limitations to a more diffuse clinical implementation of this technology. In addition, e-nose findings should be confirmed in larger series by ruling out confounders, such as comorbidities and other cancers, for which this technology has shown promising results.

      The E-Tongue and Real-Time Breath Analysis

      In addition and to complement e-nose technology, another modality of VOC detection through so-called e-tongue technology has been reported.
      • Santonico M.
      • Frezzotti E.
      • Incalzi R.A.
      • et al.
      Non-invasive monitoring of lower-limb ulcers via exudate fingerprinting using BIONOTE.
      E-tongue devices are meant to use chemometric sensors to identify compounds in liquid samples.
      • Zou Y.
      • Wan H.
      • Zhang X.
      • Ha D.
      • Wang P.
      Electronic nose and electronic tongue.
      In particular, the e-tongue consists of an electronic system controlling several electrodes immersed in a biological fluid.
      • Santonico M.
      • Frezzotti E.
      • Incalzi R.A.
      • et al.
      Non-invasive monitoring of lower-limb ulcers via exudate fingerprinting using BIONOTE.
      Several sensor arrays can be used in this system, including electrochemical, optical, mass and enzymatic sensors.
      • Zou Y.
      • Wan H.
      • Zhang X.
      • Ha D.
      • Wang P.
      Electronic nose and electronic tongue.
      Among the electrochemical sensors, the potentiometric ones measure the potential between two electrodes with no current flow as the effect of free energy change until an equilibrium status is obtained.
      • Zou Y.
      • Wan H.
      • Zhang X.
      • Ha D.
      • Wang P.
      Electronic nose and electronic tongue.
      Conversely, voltammetric sensors can measure the current produced by oxidization or reduction of redox active compounds (i.e., VOCs) with very high sensitivity.
      • Zou Y.
      • Wan H.
      • Zhang X.
      • Ha D.
      • Wang P.
      Electronic nose and electronic tongue.
      In this system, VOCs and other compounds can react with the electrodes at a certain potential given by a cyclic voltage input yielding additional information to complement the multisensorial platform used for lung cancer detection. Nevertheless, although it is theoretically worthy of assessment, the potential contribution of the e-tongue to the multisensorial detection of cancer needs further investigation.
      • Fitzgerald J.
      • Fenniri H.
      Cutting edge methods for non-invasive disease diagnosis using e-tongue and e-nose devices.
      An intriguing scenario on the use of this technology for exhaled breath fingerprinting is provided by the implementation of real-time breath analysis through proton-transfer-reaction mass spectrometry (PTR-MS), proton-transfer-reaction time-of-flight mass spectrometry (PTR-TOF-MS), or selected ion flow tube MS.
      • Haick H.
      • Broza Y.Y.
      • Mochalski P.
      • Ruzsanyi V.
      • Amann A.
      Assessment, origin, and implementation of breath volatile cancer markers.
      • Huang Z.
      • Zhang Y.
      • Yan Q.
      • Zhang Z.
      • Wang X.
      Real-time monitoring of respiratory absorption factors of volatile organic compounds in ambient air by proton transfer reaction time-of-flight mass spectrometry.
      In these systems, the gas sample interacts with excess ion charges (i.e., hydronium ions), resulting in the attendant transfer of ions to the VOCs which are then analyzed with GC-MS.
      • van der Schee M.P.
      • Paff T.
      • Brinkman P.
      • van Aalderen W.M.C.
      • Haarman E.G.
      • Sterk P.J.
      Breathomics in lung disease.
      No need for sample collection or pre-concentration procedures is contemplated with the above techniques which, however, remain far from being suitable for routine use in the clinical setting.
      • Bayrakli I.
      Breath analysis using external cavity diode lasers: a review.
      In this context, two technological developments may pave the way to the development of the entire breath analytic process in a real-time mode: matrix-assisted laser-enhanced desorption/ionization and secondary nanoelectrospray ionization MS.
      • Bayrakli I.
      Breath analysis using external cavity diode lasers: a review.
      • Li X.
      • Huang L.
      • Zhu H.
      • Zhou Z.
      Direct human breath analysis by secondary nano-electrospray ionization ultrahigh-resolution mass spectrometry: importance of high mass resolution and mass accuracy.
      These techniques are based on the concept of obtaining ionization of macromolecules contained in VOCs into analytes which are then accelerated into the MS.
      • Bayrakli I.
      Breath analysis using external cavity diode lasers: a review.
      • Li X.
      • Huang L.
      • Zhu H.
      • Zhou Z.
      Direct human breath analysis by secondary nano-electrospray ionization ultrahigh-resolution mass spectrometry: importance of high mass resolution and mass accuracy.
      A major hurdle against the wide implementation of the above techniques into clinical practice is that this instrumentation is bulky and requires complex sampling and pre-concentration methods.
      • Bayrakli I.
      Breath analysis using external cavity diode lasers: a review.
      In this setting, an important technological advancement is represented by the optical measurements of VOCs through the application of laser absorption techniques to obtain spectral fingerprints expressed in ranges of ultraviolet to mid-infrared frequencies with detection levels ranging from parts per million to parts per billion.
      • Wang C.
      • Sahay P.
      Breath analysis using laser spectroscopy techniques: breath biomarkers, spectral fingerprints, and detection limits.
      • Saidi T.
      • Moufid M.
      • Zaim O.
      • EL Bari N.
      • Ionescu R.
      • Bouchikhi B.
      Artificial sensory systems combined with UV-Vis spectrophotometry as a robust approach for VOCs analysis of human urine and exhaled breath.
      Examples of this concept are tunable diode laser absorption spectroscopy (TDLAS) and cavity ringdown spectroscopy (CRDS) with its variants (i.e., integrated cavity output spectroscopy, cavity enhanced absorption spectroscopy, and cavity leak-out absorption spectroscopy).
      • Wang C.
      • Sahay P.
      Breath analysis using laser spectroscopy techniques: breath biomarkers, spectral fingerprints, and detection limits.
      In both TDLAS and CRDS, a laser beam is passed through a gaseous environment where it is incrementally absorbed to yield a decrease of the recorded laser beam intensity.
      • Wang C.
      • Sahay P.
      Breath analysis using laser spectroscopy techniques: breath biomarkers, spectral fingerprints, and detection limits.
      In CRDS, the laser beam is injected into an optical cavity where it is continuously reflected on two opposing mirrors, thus determining the multipass nature of the optical absorption path.
      • Wang C.
      • Sahay P.
      Breath analysis using laser spectroscopy techniques: breath biomarkers, spectral fingerprints, and detection limits.
      The intensity of the light beam decays in proportion to speed of light, mirror reflectivity, and distance between mirrors; if gas is introduced in the optical cavity, the intensity of the light beam decay increases due to the gas-related absorption.
      • Wang C.
      • Sahay P.
      Breath analysis using laser spectroscopy techniques: breath biomarkers, spectral fingerprints, and detection limits.
      Conversion of the light beam signal into an acoustic pattern is the concept on which photoacoustic spectroscopy is based.
      • Wang C.
      • Sahay P.
      Breath analysis using laser spectroscopy techniques: breath biomarkers, spectral fingerprints, and detection limits.
      In brief, the acoustic waves generated by the resonance of the laser frequency interacting with the frequency of the gas molecules are detected by a sensitive microphone, thereby creating a spectral fingerprint.
      • Wang C.
      • Sahay P.
      Breath analysis using laser spectroscopy techniques: breath biomarkers, spectral fingerprints, and detection limits.
      Currently, laser spectroscopic techniques can identify several breath biomarkers and are rapidly becoming commercially available.
      • Wang C.
      • Sahay P.
      Breath analysis using laser spectroscopy techniques: breath biomarkers, spectral fingerprints, and detection limits.

      The Future of Breath Analysis: Proteomics and Neuron-Based Sensors

      The vapor phase of the breath sample can be refrigerated to 0° C or transformed into liquid through a condenser.
      • Adiguzel Y.
      • Kulah H.
      Breath sensors for lung cancer diagnosis.
      • Konstantinidi E.M.
      • Lappas A.S.
      • Tzortzi A.S.
      • Behrakis P.K.
      Exhaled breath condensate: technical and diagnostic aspects.
      The extraction of human DNA and proteins from exhaled breath condensate has been proposed for genomic and proteomic analysis.
      • Adiguzel Y.
      • Kulah H.
      Breath sensors for lung cancer diagnosis.
      • Konstantinidi E.M.
      • Lappas A.S.
      • Tzortzi A.S.
      • Behrakis P.K.
      Exhaled breath condensate: technical and diagnostic aspects.
      Compared to healthy individuals, several exhaled breath condensate biomarkers have shown variable correlation with the stage and the prognosis of the primary tumor including DNA microsatellite alterations, p53 gene mutation, increased concentrations of endothelin-1, tumor necrosis factor-α, interleukin-6, and metalloproteinase 9.
      • Konstantinidi E.M.
      • Lappas A.S.
      • Tzortzi A.S.
      • Behrakis P.K.
      Exhaled breath condensate: technical and diagnostic aspects.
      Another intriguing perspective is represented by the possibility of integrating olfactory sensory neuron-based sensors into portable e-nose devices.
      • Adiguzel Y.
      • Kulah H.
      Breath sensors for lung cancer diagnosis.
      • Son M.
      • Kim D.
      • Ko H.J.
      • Hong S.
      • Park T.H.
      A portable and multiplexed bioelectronics sensor using human olfactory and taste receptors.
      The olfactory receptors (i.e., neurons) are locked into proteins or nanotubes of the sensor array or on its surface serving as the analyte-binding component.
      • Adiguzel Y.
      • Kulah H.
      Breath sensors for lung cancer diagnosis.
      • Son M.
      • Kim D.
      • Ko H.J.
      • Hong S.
      • Park T.H.
      A portable and multiplexed bioelectronics sensor using human olfactory and taste receptors.
      By using these neuron-based sensors, the concentration threshold for detection of individual analytes can go as low as 10-12 M.
      • Adiguzel Y.
      • Kulah H.
      Breath sensors for lung cancer diagnosis.

      Conclusions

      As preconized almost 10 years ago, we may soon be witnessing a scenario in which a miniaturized, hand-held e-nose device will fit in the lab coat pocket of any clinician involved in lung cancer management, ready to be used based on the radiological suspicion of lung cancer.
      • Friedrich M.J.
      Scientists seek to sniff out diseases: electronic “noses” may someday be diagnostic tools.
      • Wilson A.D.
      • Baietto M.
      Applications and advances in electronic-nose technologies.
      Despite some obvious limitations (Table 3), the e-nose may soon become the test to guide and complement further diagnostic workup based on the evidence that it is a low-cost procedure which entails minimal distress for the patient and limited necessary logistics.
      • Wilson A.D.
      • Baietto M.
      Applications and advances in electronic-nose technologies.
      • Nardi-Agmon I.
      • Peled N.
      Exhaled breath analysis for the early detection of lung cancer: recent developments and future prospects.
      • Fitzgerald J.E.
      • Bui E.T.H.
      • Simon N.M.
      • Fenniri H.
      Artificial nose technology: status and prospects in diagnostics.
      • Wasilewski T.
      • Gębicki J.
      • Kamysz W.
      Bioelectronic nose: current status and perspectives.
      Because breathprint analysis may help predict the survival of lung cancer patients, protocols aimed at defining response to treatment and post-treatment follow-up procedures may be effectively revolutionized by the introduction of the new generations of laser-based e-nose devices and innovative sensing materials, similar to the spectroscopically encoded resins characterized in their structure by infrared and Raman spectra which are converted to disease-specific bar-codes for rapid interpretation (bar-coded resins).
      • Fitzgerald J.E.
      • Bui E.T.H.
      • Simon N.M.
      • Fenniri H.
      Artificial nose technology: status and prospects in diagnostics.
      • Schmekel B.
      • Winquist F.
      • Vikström A.
      Analysis of breath samples for lung cancer survival.
      Table 3Advantages, Limitations, And Possible Remedies of Exhalates Collecting Systems and E-Nose Devices
      LimitationsAdvantagesRemedy
      SamplingDilution of VOCs in dead space

      Lack of standardization

      (pCO2 sample / pCO2 end tidal)

      VOCs stability in bag/container/cartridge

      Storage time

      VOCs concentration

      Contamination (i.e., diet)
      For patient: easy and noninvasive procedureSemiselective sorbent materials with pre-concentration of VOCs

      Thermal desorption necessary to re-obtain VOCs

      Respiratory volumes selection

      Standardization when cartridges/containers are used instead of bags
      Pre-concentrationThermal desorption may cause VOCs degradationResolution power enhancement

      Exhaled breath separation in VOCs or VOCs families
      Microfluidic (i.e., extremely low volumes, i.e., nanoliters) management of the water component of VOCs
      GC-MS devicesSpecialized personnel

      Expensive equipment

      Sampling technique
      Provide a VOC library as referencePortable GC-MS

      GC × GC approach
      Colorimetric e-nose devicesNo VOCs identification

      Many different working principles (not univocal definition of e-nose)

      Time-consuming calibrations

      Sensor reproducibility
      Small sample size

      Sample collection is crucial
      Provide exhaled breath fingerprint

      Used in outpatient or at bedside

      No advanced training necessary

      Cost effective
      Relatively inexpensive (commercially available, electronic based)Feature selection

      Provide pattern recognition of VOCs

      Automated procedures for the covering of sensor’s surface
      Acoustic-based e-nose devicesAlterations of electrode surface coating and/or changes in humidity/temperature yieldEnhance surface coating chemosensitivity with improved materials (i.e., carbon nanotubes)
      Conductometric-based e-nose devicesNecessity of micro-heaterTemperature based selectivity
      Optical and nonoptical Spectroscopic devicesPhoto-bleaching

      Low portability
      Adjustable selectivity for specific VOCs identification

      High sensitivity
      Multiplicity of data sources (changes in intensity, fluorescence, wavelength, spectral shape)Portable spectrometer

      Arrangements/pre-treatment for sample preservation
      Compiled from various sources.
      • Haick H.
      • Broza Y.Y.
      • Mochalski P.
      • Ruzsanyi V.
      • Amann A.
      Assessment, origin, and implementation of breath volatile cancer markers.
      • Mazzone P.J.
      • Wang X.F.
      • Xu Y.
      • et al.
      Exhaled breath analysis with a colorimetric sensor array for the identification and characterization of lung cancer.
      • Wilson A.D.
      • Baietto M.
      Applications and advances in electronic-nose technologies.
      • Nardi-Agmon I.
      • Peled N.
      Exhaled breath analysis for the early detection of lung cancer: recent developments and future prospects.
      • Fitzgerald J.E.
      • Bui E.T.H.
      • Simon N.M.
      • Fenniri H.
      Artificial nose technology: status and prospects in diagnostics.
      VOC, volatile organic compound; pCO2, partial pressure of carbon dioxide; GC-MS, gas chromatography–mass spectronomy; GC × GC, comprehensive two-dimensional gas chromatography.

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