Cancers, chronic diseases and respiratory infections are major causes of mortality and present diagnostic and therapeutic challenges for health care. There is an unmet medical need for non-invasive, easy-to-use biomarkers for the early diagnosis, phenotyping, predicting and monitoring of the therapeutic responses of these disorders. Exhaled breath sampling is an attractive choice that has gained attention in recent years. Exhaled nitric oxide measurement used as a predictive biomarker of the response to anti-eosinophil therapy in severe asthma has paved the way for other exhaled breath biomarkers. Advances in laser and nanosensor technologies and spectrometry together with widespread use of algorithms and artificial intelligence have facilitated research on volatile organic compounds and artificial olfaction systems to develop new exhaled biomarkers.
The lung is an important interphase between the environment and the human body, and it serves as a major getaway for different biomolecules. Complex biological processes in different body organs have their fingerprints on exhaled breath by releasing gas phase mediators and other biomolecules that are transported to the lungs and released into the exhaled breath through the alveoli. The lung parenchyma and the airways are major sources of mediators released to the airways and make a substantial contribution to the content of exhaled breath.
1.1. The Path of Using Exhaled Volatile Compounds in Medicine
The potential of using exhaled breath to obtain information about different body functions was first recognized at the time of ancient Greek medicine when special odors were linked with different diseases such as liver cirrhosis and diabetes. It took centuries to identify and quantify the biomolecules responsible for the signals sensed by human olfaction. A landmark study was published by Pauling L et al. 
in 1971 demonstrating the presence of hundreds of volatiles in exhaled breath samples using gas–liquid partition chromatography. With the advent of gas chromatography and mass spectrometry researchers have identified and quantified thousands of volatile organic compounds (VOCs) in the breath, most of them in picomolar (10–12 mol/L or particles per trillion) concentrations 
. Different diseases have characteristic metabolic profiles that can be captured by using exhaled VOC profiles (“breathprints”). For the interpretation of huge datasets arising from a complex mixture of thousands of widely different volatile molecules to provide clinically relevant information for discrimination between health and disease and for the prediction of therapeutical responses, several statistical algorithms have been used resulting in variable levels of diagnostic accuracy 
. The large size of mass spectrometers, and the substantial expense and heavy workload required for sample processing have represented a major bottleneck for the point-of-care (POC) clinical applicability of these measurements. Two small molecules, hydrogen (H2
) and methane (CH4
), represent good examples of this transition, as they have made their way to be measured by POC tests and are widely used in the differential diagnosis of gastrointestinal disorders 
. Hydrogen and methane-based breath tests are used to diagnose and monitor small intestinal bacterial overgrowth and carbohydrate maldigestion and guide clinicians to prescribe appropriate medication 
. These tests are based on the observation that H2
are produced by the bacterial fermentation of unabsorbed carbohydrate in the small intestine during digestion and diffused to the blood that carries them to the alveoli from where they are exhaled. Since human cells do not produce them, their concentrations in breath are related to the interstitial bacterial flora.
1.2. Gaso-Transmitters in Exhaled Breath
As well as VOCs, the environmental-pollutant-free radical nitric oxide (NO), a known gaso-transmitter in the body, was also detected in exhaled breath with trace concentrations in healthy subjects and elevated levels in asthmatic patients 
. Determination of fractional exhaled NO (FeNO) has generated great interest as a potential biomarker of asthma.
This was mainly based on its correlation with eosinophils and its increase after allergen exposure, suggesting that it may be useful as a predictive marker of asthma attacks and the therapeutic response 
. FeNO has served as a prototype of exhaled biomarkers for disease monitoring and medical decision making. Several machines have US Food and Drug Administration approval and/or a European Union CE-mark as medical device for its measurement 
. The other two toxic environmental pollutants with known gaso-transmitter functions in the human body, carbon monoxide (CO) and hydrogen sulfide (H2
S), can also be detected in exhaled breath. Their levels are altered in different diseases, such as asthma, chronic obstructive pulmonary disease (COPD) and cystic fibrosis 
. However, the use of exhaled CO as a biomarker of heme oxygenase activity is hampered by the strong and long-standing effect of smoking on the exhaled CO level, and the exhaled H2
S level is profoundly influenced by its oral and gastrointestinal bacterial production 
1.3. Biological and Artificial Olfaction Systems to Assess Exhaled Volatiles
Several species have a lot more sensitive olfactory systems than humans including dogs, rats and different insects. The specific coupling of large numbers of receptors with the brain neural network enables these species to recognize minor changes in the volatome of different human samples including the breath. This led to the idea of involving trained animals in human medicine and diagnostics. Sniffer dogs have been trained successfully to distinguish biological samples obtained from healthy and diseased individuals. They have been shown to identify patients with Parkinson’s disease 
, lung cancer 
, prostate cancer 
, ovarian cancer 
and different infectious diseases 
from samples such as urine, blood, serum, cell lines and bacterial cultures with very high sensitivity. Dogs can also be trained to alert to hypoglycemic periods in type 1 diabetics 
. Moreover, even untrained dogs have been shown to sniff out the prodromal phase of seizures and respond to the unusual odor changes with an increase in affiliative behaviour directed at their owners 
. As well as dogs, other animal species have been tested in odor-pattern-based diagnostics. For instance, African giant pouched rats can detect Mycobacterium tuberculosis
, the pathogens causing tuberculosis very sensitively 
, and are indeed used for first line diagnostics in Africa. Insects, such as mosquitos and honeybees also have a very sensitive olfaction with great discriminatory power to detect a tremendous amount of chemical signals 
. Moreover, bees have already been successfully trained to detect specific odors 
Compared to the detection and quantification of individual molecules, using the biological olfactory systems of animals as a model to build artificial olfaction systems, so-called electronic noses, is a completely different approach. Electronic noses consist of arrays of chemical vapor sensors that respond to certain characteristics of odorant molecules including exhaled VOCs. Sensors are not specific to a given molecule, a sensor may react with several different molecules and a given molecule may also generate responses from several sensors. In this approach individual molecules are not identified and quantified as they are by mass spectrometry; only the pattern of sensor responses (“breathprint”) induced by a complex mixture of different volatiles is clustered. Despite the limitation of the black box approach due to the versatile nature of potential arrays of chemosensitive sensors, their small size and low cost, they have gained great attention as potential point-of-care clinical tools 
. Their integration with artificial intelligence for data analysis has contributed importantly to the rapid development of this field 
2. Exhaled Gaso-Transmitters
There are three known gaso-transmitters in the human body: NO, CO and H2
S. They are widely different molecules. They are all counted as environmental pollutants and toxic gases. As bioactive molecules they have important anti-inflammatory, antioxidative, antiproliferative and antiapoptotic properties, and their low-dose inhalation or administration of their donor molecules can provide therapeutic effects in different conditions 
. Due to their environmental occurrence, when their levels are measured in exhaled breath, special attention is required to limit the potential environmental influence. This is a complex task because it is not enough to determine the background environmental levels as environmental gases once inhaled could stay in the human body for different time lengths that depends on their physicochemical nature. They either could be exhaled immediately, or they might pass the alveolo-capillary membranes and circulate in the body for several hours and be added to exhaled breath in later breathing cycles 
. They interact with different molecules, and in this way, they can be transformed into other molecules that may result in lower than environmental concentrations in exhaled breath. The other methodological challenge is that their bodily production and transportation results in very low concentrations being present in exhaled breath, requiring very sensitive detection systems.
3. Exhaled Hydrogen Peroxide
is an oxygen metabolite that diffuses through cells and tissues and serves important metabolic and regulatory roles under physiological and pathophysiological circumstances. It is an important signaling molecule playing a part in cellular adaptation to environmental stress as a part of redox signaling pathways 
. In oxidative stress and inflammation, NO, CO and H2
S are interrelated with H2
and other reactive oxygen species in multiple ways 
. Exhaled H2
can be captured in exhaled breath condensate (EBC), a cooled breath sample containing large numbers of volatile and non-volatile biomaterials 
. The level of exhaled H2
is extremely variable and depends on several factors that having a direct or indirect influence on its level. Environmental conditions, ventilatory pattern, measurement techniques and storage influence its concentration directly, but they may also act indirectly by changing the pH of EBC 
. To limit variability due to sample storage and support point-of-care detection, different online detection systems and disposable sensors have been built and tested 
To allow deeper understanding of oxidative-stress-related processes and interactions between different mediators, micromachines able to detect complete sets of molecules from the same sample are desirable.
4. Breathomics—Breath Fingerprinting
Different diseases have characteristic metabolic profiles that can be captured by using metabolomics, proteomics and other “omic” technologies in different biological samples. Using “omics” for biomarker discovery studies is one of the important pathways enabling us to reconstruct our understanding of different chronic diseases by measuring exhaled breath volatiles 
. Thousands of different VOCs have been detected in exhaled breath that can be identified and quantified by mass-spectrometry-based methodologies or samples that can be discriminated based on the patterns by electronic or biological noses 
. Exhaled VOCs are principally isoprene, alkanes, methylalkanes and benzene derivatives. They are related to widely different cellular functions and metabolic processes including lipid peroxidation, oxidative stress and cholesterol synthesis among others 
. Endogenously produced VOCs can be detected in different samples, such as exhaled breath, urine, feces, saliva and blood. The concentration of a given VOC in exhaled breath is also influenced by alveolar minute ventilation and cardiac output together with its blood–gas partition coefficient. As well as endogenous formation, they can also be found in the environment or in other exogenous sources (food and drink, diagnostic test drugs, medication, smoking, etc.). VOCs found in biological samples cannot, therefore, solely reflect bodily functions because exogenous VOCs also have an influence on the exhaled samples. Discrimination between the two sources in exhaled breath samples is challenging and relies on using different breathing maneuvers, assessing the effect of VOC clean gases for inhalation, using filters in the inhalation loop of the sampling device and keeping a certain time gap between exposure and sampling (i.e., subject is requested not to smoke for 1–12 h before sample collection). A specific potential confounding source is the collecting device itself because several materials and most cleaning fluids release VOCs, and that is extremely hard to exclude. In general, environmental influence on the concentration of exhaled VOCs cannot be completely ruled out by any of the currently used approaches 
Various sampling and analytical methods have been used to assess the metabolome through exhaled breath. While individual gaso-transmitters paved the way for clinically useful point-of-care measurements, currently, the rapid development in sensor technology and the application of artificial intelligence have resulted in major developments in the field of breathomics, a promising field for easy-to-use, point-of-care machines for diagnostic and monitoring purposes. Advanced wearable sensors to detect biomolecules in fluids or exhaled breath open a way for potential online home monitoring 
. The main areas of interest are screening, diagnosis, phenotyping, exacerbation prediction, exacerbation etiology and prediction of the treatment response where a major breakthrough can be achieved with the envisioned micromachines.