Development of a new portable device designed for selective olfaction

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A mia sorella Gaia, e ai miei genitori

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FACOLT `A DI INGEGNERIA

CORSO DI LAUREA SPECIALISTICA IN INGEGNERIA

BIOMEDICA

Anno accademico 2013/14

Tesi di Laurea Specialistica

Development of a new portable device designed for

selective olfaction

Relatori

Candidata

Ing. Andrea Ginghiali

Danila Germanese

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Introduction

Human breath is largely composed of oxygen, carbon dioxide, water vapor, nitric oxide, and numerous volatile organic compounds (VOCs). The type and number of the VOCs in the breath of any particular individual will vary but there is nonetheless a comparatively small common core of breath which is present in all humans.

To date, more than 1000 molecules and different compounds have been iden-tified to be present in exhaled human breath. Among these:

• molecules or their metabolites originating from inhaled air, from dermal absorption, from foods and beverages (exogenous molecules);

• molecules produced by anabolic or catabolic reactions that occur in tissues or cells throughout the body. These molecules, that are en-dogenous, are present in breath relative to their types, concentrations, volatilities, lipid solubility, and rates of diffusion as they circulate in the blood and cross the alveolar membrane.

Approximately, 35 of the identified compounds in the exhaled breath have been established as biomarkers for particular diseases and metabolic disor-ders. It means, changes in the concentration of the molecules in these VOCs could suggest various diseases or, at least, changes in the metabolism. Such biomarkers have been identified using instrumentations such as gas

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expensive and not portable, its sampling and assaying processes are compli-cated and time consuming (about one hour for one sample), and its results require expert interpretation. E-nose is cheaper and faster (requiring only 30 minutes for one sample) but it is often used outside of medicine, in fields related to food, chemistry, fragrances, security, and environment. Recently, e-nose has gradually been used in medicine for the diagnosis of renal disease, diabetes, lung cancer, and asthma, but it can identify only one particularv disease. One reason for the limited applications of e-noses in breath analysis might be the design of commercial e-noses for broad applications rather than for breath analysis specifically.

As a consequence, in recent years, the necessity to develop a portable device for breath analysis, easy to use, and feasible for patients living far from med-ical structures or physicians.

In this work, we describe the design and functionality of the first prototype of a new portable device for breath analysis limited to an effective number of substances, the so called Wize Sniffer 1.0. My basic original contribu-tion to this thesis work, carried out at the Institute of Informacontribu-tion Science and Technologies (ISTI) of CNR, and started on Semptember 2013, regarded:

• the design of the hardware platform, including the Arduino Ethernet board, the circuit for the connection between Arduino board and an array of commercial gas sensors;

• the communication protocol between sensors and the notebook (laptop) both via serial cable and wireless;

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• data analysis.

The idea of the Wize Sniffer takes place within SEMEOTICONS (SEMEiotic Oriented Technology for Individual’s CardiOmetabolic risk self-assessmeNt and Self-monitoring, www.semeoticons.eu), an European triennial project started on November 2013 whose central idea is to exploit the face as a major indicator of individual’s well-being and to assess a num-ber of computational descriptors to atherosclerotic cardiovascular diseases that are leading causes of mortality worldwide. These descriptors can be ex-pressed involving (i) morphometric, biometrics and colorimetric of the face; (ii) spectroscopic analysis of skin and iris, of sub-cutaneous substances and the function of subcutaneous tissues, and (iii) compositional analysis of ex-haled breath.

Such analysis will be made through an innovative multisensory system inte-grated into a hardware platform having the exterior aspect of a mirror: the so-called ”Wize Mirror”.

Within the SEMEOTICON Project by the Wize Sniffer, we intend a hard-ware/software tool for the analysis of volatile organic compounds of breath, to be integrated in the Wize Mirror. The Wize Sniffer should be able to pro-vide useful information about the ”breathprint”, the analog of fingerprint for the state of health of an individual, and to give, in case, feedbacks about alcohol intake and smoking habit. For this purpose, as described also in [3], the Wize Sniffer 1.0. should be able to detect carbon monoxide (the major component present in tobacco fumes), hydrogen (resulting from carbohy-drate fermentation by anaerobic bacteria), ethanol (endogenous, originated from microbial fermentation of carbohydrates in the gastro-intestinal tract, and exogenous, deriving from alcoholic drink), ammonia (another compo-nent present in tobacco fumes), carbon dioxide (which production can be

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The hardware design of the Wize Sniffer 1.0. is described, as well as the soft-ware design. About the hardsoft-ware design, we took inspiration from a study, conducted by D. Guo et al. [2], whose aim was to develop a new specific breath analysis system making use of chemical sensors that were particularly sensitive to the biomarkers and compositions in human breath. They used metal oxide semiconductor gas sensors, manufactured by Figaro Enginnering. Being these sensors very robust, sensitive, resistant to humidity and ageing, easy to read, we retained very reasonable using this kind of sensors.

The box embedding the sensors was designed in collaboration with COSMED s.r.l. Its general scheme had a gas store bag/box (A), an element (B) whose task was to absorb the water vapor from the breath, and a disposable mouth-piece (C). The gas sensors, placed in the store chamber, sense gas particles and generate measurable electronic signals. The data from the sensors are subsequently sent to the computer by means of TCP/IP trasmission protocol and saved for further analysis.

In order to evaluate Wize Sniffer 1.0.’s performances and make it ready to be integrated in the ”Wize Mirror”, we drafted a measuring protocol to test our device on a population containing individuals of different age, habits, body type.

In the conclusive section of this work, the test results are discussed, as well as the advantages and the limits of our device compared with the traditional breath analysis techniques. A ”to do list”, for Wize Sniffer future develop-ments, is drafted, in order to improve its performances.

Indeed, in order to obtain higher sensitivity, the integration of polyaniline electrospun nanofibers as sensors’ sensing element, will be the next great challenge.

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Chapter 1 Something about breathing

1.1 Human respiratory system

The respiratory tract starts at the nose and mouth, goes ahead with phar-ynx, larynx and trachea, and stops with bronchi, which run into lungs, before branching off into smaller secondary and tertiary bronchi, bronchioles and alveoli.

The nose form the main external opening for the respiratory system and is the first section of the body through which air moves. The function of the nasal cavity is to warm, moisturize, and filter air entering the body before it reaches the lungs. Hairs and mucus lining the nasal cavity help to trap dust, mold, pollen and other environmental contaminants before they can reach the inner portions of the body. Air exiting the body through the nose returns moisture and heat to the nasal cavity before being exhaled into the environment.

The oral cavity is the secondary external opening for the respiratory tract. Most normal breathing takes place through the nasal cavity, but the oral cav-ity can be used to supplement or replace the nasal cavcav-ity’s functions when

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Figure 1.1: A schematic view of human respiratory system

needed. Because the pathway of air entering the body from the mouth is shorter than the pathway for air entering from the nose, the mouth does not warm and moisturize the air entering the lungs as well as the nose performs this function. The mouth also lacks the hairs and sticky mucus that filter air passing through the nasal cavity. The one advantage of breathing through the mouth is that its shorter distance and larger diameter allows more air to quickly enter the body.

The pharynx, also known as the throat, is a muscular funnel that extends from the posterior end of the nasal cavity to the superior end of the esopha-gus and larynx. The pharynx is divided into three regions: the nasopharynx, oropharynx, and laryngopharynx. Inhaled air from the nasal cavity passes into the nasopharynx, descends through the oropharynx, end then into the laryngopharynx, where it is diverted into the opening of the larynx by the epiglottis. The epiglottis is a flap of elastic cartilage that acts as a switch between the trachea and the esophagus.

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1.1. HUMAN RESPIRATORY SYSTEM

The larynx connects the laryngopharynx and the trachea. It contains also special structures known as vocal folds, which allow the body to produce the sounds of speech and singing. The vocal folds are folds of mucous membrane that vibrate to produce vocal sounds. The tension and vibration speed of the vocal folds can be changed to change the pitch that they produce. The trachea connects the larynx to the bronchi and allows air to pass into the thorax. The rings of cartilage making up the trachea allow it to remain open to air at all times. The main function of the trachea is to provide a clear airway for air to enter and exit the lungs. In addition, the epithelium lining the trachea produces mucus that traps dust and other contaminants and prevents it from reaching the lungs.

At the inferior end of the trachea, the airway splits into left and right branches known as the primary bronchi. The left and right bronchi run into each lung before branching off into smaller secondary bronchi. The secondary bronchi carry air into the lobes of the lungs. The secondary bronchi in turn split into many smaller tertiary bronchiwithin each lobe. The tertiary bronchi split into many smaller bronchioles that spread throughout the lungs. Each bronchiole further splits into many smaller branches less than a millimeter in diameter called terminal bronchioles. Finally, the millions of tiny terminal bronchioles conduct air to the alveoli of the lungs.

The main function of the bronchi and bronchioles is to carry air from the trachea into the lungs. Smooth muscle tissue in their walls helps to regulate airflow into the lungs. When greater volumes of air are required by the body, such as during exercise, the smooth muscle relaxes to dilate the bronchi and bronchioles. The smooth muscle fibers are able to contract during rest to prevent hyperventilation. The bronchi and bronchioles also use the mucus and cilia of their epithelial lining to trap and move dust and other

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contami-nants away from the lungs.

Each lung is surrounded by a pleural membrane that provides the lung with space to expand as well as a negative pressure space relative to the body’s exterior. The negative pressure allows the lungs to passively fill with air as they relax. The interior of the lungs is made up of spongy tissues containing many capillaries and around 30 million tiny sacs known as alveoli. The alveoli are cup-shaped structures found at the end of the terminal bronchioles and surrounded by capillaries. The alveoli are lined with thin simple squamous epithelium that allows air entering the alveoli to exchange its gases with the blood passing through the capillaries.

The principal muscle of respiration in the human body is the diaphgram. When the diaphragm contracts, it moves inferiorly into the abdominal cavity, expanding the space within the thoracic cavity and pulling air into the lungs. Relaxation of the diaphragm allows air to flow back out the lungs during ex-halation. Between the ribs are many small intercostal muscles that assist the diaphragm with expanding and compressing the lungs. These muscles are divided into 2 groups: the internal intercostal muscles and the external intercostal muscles. The internal intercostal muscles depress the ribs to com-press the thoracic cavity and force air to be exhaled from the lungs. The external intercostals elevate the ribs, expanding the volume of the thoracic cavity and causing air to be inhaled into the lungs.

1.2 Breathing mechanism

Breathing is the movement of air from outside the body into the bronchial tree and alveoli, followed by a reversal of this air movement. The actions

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1.2. BREATHING MECHANISM

responsible for these air movements are termed inspiration, or inhalation, and expiration, or exhalation.

Inspiration

Atmospheric pressure due to the weight of the air is the force that moves air into the lungs. Normal air pressure equals 760mm of mercury (Hg) (= 1 atmosphere).

When the respiratory muscles are at rest, the pressures on the inside of the lungs and alveoli and on the outside of the thoracic wall are about the same. Pressure and volume are related in an opposite, or inverse, way. If the pres-sure inside the lungs and alveoli (intra alveolar prespres-sure) decreases, outside air will then be pushed into the airways by atmospheric pressure. This is what happens during normal inspiration, and it involves the action of muscle fibers within the diaphragm.

The muscle fibers of the diaphragm are stimulated to contract by impulses carried by the phrenic nerves which are associated with the cervical plexuses. When this occurs, the diaphragm moves downward, the thoracic cavity en-larges, and the intra-alveolar pressure falls about 2mm below that of atmo-spheric pressure. In response, air is forced into the airways by atmoatmo-spheric pressure.

While the diaphragm is contracting and moving downward, the external (in-spiratory) intercostal muscles and certain thoracic muscles may be stimulated to contract.

This action raises the ribs and elevates the sternum, increasing the size of the thoracic cavity even more. As a result, the intra-alveolar pressure falls farther, and atmospheric pressure forces more air into the airways.

If a person needs to take a deeper than normal breath, the diaphragm and external intercostal muscles contract more forcefully. Additional muscles,

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such as the pectoralis minors and sternocleidomastoids, can also be used to pull the thoracic cage further upward and outward, enlarging the thoracic cavity, and decreasing pressure even more.

The ease with which the lungs can expand as a result of pressure changes occurring during breathing is called compliance (distensibility). In a normal lung, compliance decreases as lung volume increases , because an inflated lung is more difficult to expand than a lung at rest.

Expiration

The forces responsible for normal resting expiration come from elastic recoil of lung tissues. When the diaphragm lowers, the abdominal organs inferior to it are compressed. As the diaphragm and the external intercostal muscles relax following inspiration, the elastic tissues cause the lungs to recoil, and they return to their original shapes. Similarly, elastic tissues within the ab-dominal organs cause them to spring back into their previous shapes, pushing the diaphragm upward.

These factors increase the intra - alveolar pressure about 1 mm Hg above atmospheric pressure , so the air inside the lungs is forced out through the respiratory passages.

Because normal resting expiration occurs without the contraction of muscles, it is considered a passive process.

1.3 Respiratory volumes and capacity

Different degrees of effort in breathing move different volumes of air in or out of the lungs.

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1.3. RESPIRATORY VOLUMES AND CAPACITY

The volume of air that enters or leaves during a respiratory cycle is termed the tidal volume. About 500ml - 600ml of air enter during a normal, resting inspiration. On the average, the same volume leaves during a normal, resting expiration. Thus, the resting tidal volume is about 500 ml.

During forced maximal inspiration, a volume of air in addition to the resting tidal volume enters the lungs. This additional volume is called the inspira-tory reserve volume (complemental air), and it equals about 3000 ml. During a maximal forced expiration, about 1100 ml of air in addition to the resting tidal volume can be expelled from the lungs; this volume is called the expiratory reserve volume (supplemental air).

Once the respiratory volumes are known, four respiratory capacities can be calculated by combining two or more of the volumes. If the inspiratory re-serve volume (3000 ml) is combined with the tidal volume (500 ml) and the expiratory reserve volume (1100 ml), the total is termed the vital capacity (4600 ml). This capacity is the maximum volume of air a person can exhale after taking the deepest breath possible.

The tidal volume (500 ml) plus the inspiratory reserve volume (3000 ml) gives the inspiratory capacity (3500 ml), which is the maximum volume of air a person can inhale following a resting expiration. Similarly, the expira-tory reserve volume (1100 ml) plus the residual volume (1200 ml) equals the functional residual capacity (2300 ml), which is the volume of air that remains in the lungs following a resting expiration. The vital capacity plus the residual volume equals the total lung capacity (about 5800 ml). This, obviously, varies with age, sex, and body size.

Some of the air that enters the respiratory tract during breathing fails to reach the alveoli. This volume (about 150 ml) remains in the passageways of the trachea, bronchi, and bronchioles. It is called dead space air and it

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equals about 150 ml.

The volume of new atmospheric air that is moved into the respiratory pas-sages each minute is called the minute ventilation. It equals the tidal volume multiplied by the breathing rate. Thus, if the tidal volume is 500 ml and the breathing rate is 12 breaths per minute, the minute ventilation is 500mL x 12= 6000 ml min−1. However, much of the new air remains in the physiologic dead space.

The volume of new air that does reach the alveoli and is available for gas exchange is calculated by subtracting the physiologic dead space (150 ml) from the tidal volume (500 ml). The resulting volume (350 ml) multiplied by the breathing rate (12 breaths per minute) is the alveolar ventilation rate (4200 ml min−1), which affects the concentrations of oxygen and carbon dioxide in the alveoli.

A number of factors influence breathing rate and depth.

These include Oxygen partial pressure PO₂ and Carbon Dioxide partial pres-sure PCO₂ in body fluids, the degree to which lung tissues are stretched, and emotional state. The receptors involved include mechanoreceptors as well as central and peripheral chemoreceptors.

These chemoreceptors respond to changes in blood pH that correspond to variation in CO₂ partial pressure in plasma.

In any event, breathing rate and tidal volume increase when a person inhales air rich in carbon dioxide or when body cells produce excess carbon dioxide. These changes increase alveolar ventilation. As a result, more carbon diox-ide is exhaled, and the blood PCO₂ and hydrogen ion concentration return toward normal.

When PO₂ decreases the breathing rate and tidal volume increase, thus in-creasing alveolar ventilation. This mechanism does not usually play a major

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1.3. RESPIRATORY VOLUMES AND CAPACITY

role until the PO₂ decreases to about 50% of normal; thus, oxygen plays only a minor role in the control of normal respiration.

The limited role of PO₂ may be surprising, considering the importance of oxygen for sustaining life. Because most blood oxygen is carried on the hemoglobin in red blood cells, deoxygenated systemic venous blood still has 75% of the oxygen it had when it was fully oxygenated. This large excess of oxygen ”trees up” respiratory control from paying attention to blood oxygen levels under most circumstances.

Thus, the respiratory system can focus on blood PCO₂ which is important in maintaining the pH of the internal environment. Emotional upset or strong sensory stimulation may alter the normal breathing rate. Gasping and rapid breathing are familiar responses to fear, anger, shock, excitement, horror, surprise.

Moreover, because control of the respiratory muscles is voluntary, we can alter breathing pattern consciously, or even stop it for a short time. If a person decides to stop breathing, the blood concentrations of carbon dioxide begin to rise, and the concentration of oxygen falls. On the other hand, a person can increase the breath- holding time by breathing rapidly and deeply in advance. This action, termed hyperventilation, lowers the blood carbon dioxide concentration below normal.

Exercise also increases respiratory rate, due to the action of proprioceptors, the increase in body temperature, the release of epinephrine, and motor im-pulses originating from the brain.

Anyway, if a correct ventilation is not maintained, two opposing conditions could occur: respiratory acidosis, a life threatening condition, and respira-tory alkalosis.

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Figure 1.2: The respiratory tubes end in tiny alveoli, each of which is sur-rounded by a capillary network

1.4 Gas exchanges

The alveoli are the sites of the vital process of gas exchange between the air and the blood (see Figure 1.2). The exchange occurs through respiratory membrane (alveolar-capillary membrane).

Molecules diffuse from regions where they are in higher concentration to-ward regions where they are in lower concentration (diffusion process). Thus, in determining the direction of diffusion of a solute, we must know the con-centration gradient, as postulated by first Fick’s law:

J = −Ddθ

dx (1.1)

where:

• J (mol/m2_{sec)is the ”diffusion flux”; it means, it measures the amount}

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1.4. GAS EXCHANGES interval

• D is the diffusion coefficient (m2_/s)

• θ is the concentration (mol/m3₎

• x isthe position (m)

D is proportional to the squared velocity of the diffusing particles, which de-pends on the temperature, viscosity of the fluid and the size of the particles. For biological molecules the diffusion coefficients normally range from 10−11 to 10−10 m2_{/s. In the case of gases, it is more convenient to think in terms}

of a partial pressure gradient. When a mixture of gases dissolves in blood, the resulting concentration of each dissolved gas is proportional to its partial pressure. Each gas diffuses between blood and its surroundings from areas of higher partial pressure to areas of lower partial pressure until the partial pressures in the two regions reach equilibrium.

For example, the PCO₂of blood entering the pulmonary capillaries is 45mmHg, but the PCO₂ in alveolar air is 40 mmHg. Because of the difference in these partial pressures, carbon dioxide diffuses from blood, where its partial pres-sure is higher, across the respiratory membrane and into alveolar air. When blood leaves the lungs, its PCO₂ is 40 mmHg which is the same as the PCO₂ of alveolar air (see Fig. Figure 1.3(a) and Fig. Figure 1.3(b) )

Similarly, the PO₂ of blood entering the pulmonary capillaries is 40 mmHg, but reaches 104 mmHg as oxygen diffuses from alveolar air into the blood. Thus, since equilibrium is reached, blood leaves the alveolar capillaries with a PO₂ of 104 mmHg (some venous blood draining the bronchi and bronchioles mixes with this blood before returning to the heart, so the PO₂ of systemic arterial blood is 95 mmHg). Because of the large volume of air always in the lungs, as long as breathing continues, alveolar PO₂ stays relatively constant

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(a) (b)

Figure 1.3: Oxygen diffuses from the air within the alveolus into the capil-lary,while carbon dioxide diffuses from the blood within the capillary into the alveolus.Gases are exchanged because of differences in partial pressures.

at 104 mmHg.

The respiratory membrane is normally so thin that certain soluble chemicals other than carbon dioxide may diffuse into alveolar air and be exhaled. This is why breath analysis can reveal alcohol in the blood or acetone can be smelled on the breath of a person who has untreated diabetes mellitus. Breath analysis may also detect substances associated with kidney failure, certain digestive disturbances, and liver disease.

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Chapter 2 Breath analysis: state of the art

Since the time of Hippocrates, classical medicine has used ”subjective im-pressions” of the odors of the body to suggest diagnoses: fruit like scent of acetone, underlying diabetes, or the fumes of urine, indicating kidney dis-eases.

In 1784, Lavoisier, in collaboration with Laplace, first detected the pres-ence of Carbon Dioxide in human breath.

However, it is generally accepted that modern breath analysis started with the discovery, made by Pauling in 1971, that hundreds of volatile organic compounds (VOCs) are present in normal human breath at the levels of parts per billion (ppb) or lower. This discovery, together with other early breath studies, has generated research interest in human breath analysis for non-invasive disease diagnosis, metabolic status and progress of therapies monitoring.

Comparing with other traditional methods such as blood and urine test, breath analysis is non-invasive, real-time, and least harmless to not only the subjects but also the personnel who collect the samples.

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metabolites after administration of a drug or substrate, and analysis of breath compounds produced endogenously due to a particular physiological status. Our interest has been focused on endogenous VOCs, which are described in the following section.

An overview on the different methods for the analysis of human exhaled gas will complete our knowledge about the state of art.

2.1 Molecules and Volatile Organic Compounds

(VOCs) present in human breath

Human breath is largely composed of oxygen, carbon dioxide, water vapor, nitric oxide, and numerous volatile organic compounds (VOCs). The type and number of the VOCs in the breath of any particular individual will vary but there is nonetheless a comparatively small common core of breath which are present in all humans.

To date, more than 1000 molecules and different compounds have been iden-tified to be present in exhaled human breath. Among these:

• molecules or their metabolites originating from inhaled air, from dermal absorption, from foods and beverages (exogenous molecules);

• molecules produced by anabolic or catabolic reactions that occur in tissues or cells throughout the body. These molecules, that are en-dogenous, are present in breath relative to their types, concentrations, volatilities, lipid solubility, and rates of diffusion as they circulate in the blood and cross the alveolar membrane.

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2.1. MOLECULES AND VOLATILE ORGANIC COMPOUNDS (VOCS) PRESENT IN HUMAN BREATH

Their concentrations range from ppb to ppt levels.

Approximately, 35 of the identified compounds in the exhaled breath have been established as biomarkers for particular diseases and metabolic disor-ders. It means, changes in the concentration of the molecules in these VOCs could suggest various diseases or, at least, changes in the metabolism. All of these biomarkers and their related diseases and metabolic disorders are listed in Figure 2.1.

Formation of these compounds is attributed to the biochemical reactions happening inside the body as a part of metabolic pathway. Unfortunately, detailed formation mechanisms of all of the biomarkers and how each of them is related to a specific disease are not well understood. In some cases, a breath species is a biomarker that is indicative of about more than one disease or metabolic-disorder; in other cases, one particular disease or metabolic disor-der can be characterized by more than one chemical species. Moreover, the onset of a disease results in changes in the concentrations of breath molecules and not the production of unique breath biomarkers.

For example, breath nitric oxide ( NO ) is a biomarker for asthma, bronchieac-tasis, and rhinitis; Ethane is a biomarker for vitamin E deficiency in children and an indicator of lipid peroxidation, and it provides also as a bio-marker for asthma. At the same time, hydrogen peroxide ( H₂O₂ ) and ammonia ( NH₃ ) are also biomarkers for asthma. Similarly, lipid peroxidation can be diagnosed through analysis of breath ethylene and pentane. Moreover, pentane together with carbon disulfide is a biomarker of schizophrenia. As a consequence, breath analysis has to be not only highly-sensitive but also highly-selective in order to obtain accurate information.

Let’s go and see in detail some of these biomarkers present in our exhaled breath.

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Figure 2.2: Generation of Acetone via decarboxylation of acetyl-CoA

Acetone(CH₃-CO-CH₃)

Among all of volatile organic compounds (VOCs) and gases in the human breath, acetone is one of the most abundant.

The mean acetone concentration in healthy breath reported in the litera-ture varies from 0,39 ppm to 0,85 ppm, and the overall mean value is about 0,49 ppm

Acetone is considered a good indicator for monitoring fat metabolism. In a previous study ([10]) it was found that acetone contained in our exhaled breath is a metabolic product of the breakdown of body fat.

It is generated through decarboxylation of acetoacetate produced through condensation of Acetyl-CoA (which itself comes from βoxidation of fatty acids).

Moreover, in the same study, it was found that body fat, in subjects with a controlled caloric intake and taking exercise, decreased significantly, whereas breath acetone concentrations in those subjects increased significantly. In addition, acetone values increased during prolonged fasting and decreased

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after a protein-rich meal.

According to another study ([5]), in cases of severe diabetics patients (espe-cially those who have diabetes of type 1), metabolism of fats is increased, resulting in a lack of oxyloacetate, a subsequent buildup of acetyl-CoA and a progressive increasing acetone concentration together with blood glucose concentration. Naturally, acetone is produced in the blood, and since there is a strong correlation between the concentration of breath acetone and that of blood acetone, breath analysis could be very attractive for diabetics who have to control routinely their blood glucose level.

Acetone concentrations in human breath can also be an indicator of conges-tive heart failure and cardiac index. In a study ([4]) intended to assess the impact of heart surgery with extracorporeal circulation (ECC) onto breath biomarker profiles, special attention was attributed to oxidative or metabolic stress which can cause organ damage. Acetone concentrations were analyzed to investigate the effects of surgery and ischemia/reperfusion onto dextrose and cholesterol metabolism, metabolic stress and lipolysis. It was found that exhaled acetone concentrations increased slightly after sternotomy and markedly after end of ECC. This can be explained with a release of cate-cholamines that results in an increased lipolysis, especially when levels of catecholamines are artificially raised in order to wean the patient from ECC. The same holds true for the presence of other inflammation which can cause an increased need for endogenous or therapeutically applied catecholamines. In conclusion, acetone in breath could be considered non only a biomarker for monitoring fat metabolism, but also a biomarker for metabolic stress. As this volatile biomarker can be determined easily, exhaled acetone could be used to recognize these pathological conditions and to prevent patients from being damaged by them.

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Figure 2.3: Production of ammonia by amino acids deamination and its con-version into urea in liver

Ammonia(NH₃)

Mean ammonia concentration in exhaled breath of healthy subjects varies in a range of 425-1800 ppb, with mean 960 ppb.

Ammonia is a metabolic product of amino acids deamination, catalyzed by enzymes such as glutamate dehydrogenase 1.

It is toxic in high concentrations, that is why it is converted in urea (much less toxic) in liver (see Figure 2.3).

According to a study ([9]) significant levels of ammonia will appear in the blood if the removal of ammonia through conversion to urea is limited due to an impairment of liver function (as in case of cirrhosis).

As a consequence, elevated breath ammonia could be due to liver disease, but also to kidney disease (there could be an uremia, that is an inability of the kidneys to effectively filter the blood, resulting in a build up of nitrogen based compounds), genetic diseases, and also periodontal disease.

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uremia, was approximately 4880 ppb with a range of 820-14 700 ppb.

Ammonia is also one of the three major compounds, together with nitric oxide and carbon monoxide, of tobacco fumes (it is present in approximately 22,15%). All these three gases can have detrimental effects on the respira-tory system. About ammonia, it forms a strong alkaline solution with water. When inhaled, ammonia irritates the upper respiratory tract including the nasal cavity, pharynx, larynx and trachea. Irritation results in coughing and hoarseness.

Carbon monoxide(CO)

Mean carbon monoxide concentration in exhaled breath is about 3,5 ppm. Naturally, carbon monoxide presents in human breath is related to the one present in blood. It is produced by the action of heme oxygenase on heme, as shown in Figure 2.4. Heme degradation begins inside macrophages of the spleen, which remove old and damaged (senescent) erythrocytes from the circulation. In the first step, heme is converted to biliverdin by the enzyme heme oxygenase (HMOX). NADPH is used as the reducing agent, molecular oxygen enters the reaction, carbon monoxide (CO) is produced and the iron is released from the molecule as the ferric ion Fe₃+.

CO acts as a cellular messenger, as a promoter of neurovascular growth (it’s not always beneficial, since it plays a bad role in tumor growth) and func-tions in vasodilation. Because of these carbon monoxide’s roles in the body, abnormalities in its metabolism have been linked to a variety of diseases, in-cluding neurodegenerations, hypertension, heart failure, and inflammation. Moreover, heme degradation appears to be an evolutionary conserved re-sponse to oxidative stress. Briefly, when cells are exposed to free radicals, there is a rapid induction of the expression of the stress responsive heme

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Figure 2.4: Production of carbon monoxide by action of heme oxygenase on heme

oxygenase-1 (HMOX1) isoenzyme that catabolizes heme. The reason why cells must increase exponentially their capability to degrade heme in response to oxidative stress remains unclear but this appears to be part of a cytopro-tective response that avoids the deleterious effects of free heme.

Carbon monoxide is also absorbed through breathing and enters the blood stream through gas exchange in the lungs.

Normal circulating levels in the blood are 0% to 3%, and are higher in smok-ers. Indeed, carbon monoxide is the major component present in tobacco fumes (75,95%).

So, increasing levels of exhaled carbon monoxide can be detected in smoking subjects: 13.8 - 29 ppm.

An increase of CO leads hemoglobin to carry less oxygen through the vessels, because CO usurps the space in hemoglobin that normally carries oxygen, forming carboxyhemoglobin. Amounts of oxygen that is lower than 87% -97% (normal levels) may result in hypoxia.

In conclusion, carbon monoxide can be considered a biomarker for oxida-tive stress, for respiratory inflammations, for anemia, and potentially for atherosclerosis issues.

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Carbon dioxide(CO₂)

Mean carbon dioxide concentration in exhaled breath is about 4% (= 40000ppm). It is produced as a waste product when energy is released during certain metabolic reactions of cellular respiration, as shown in Figure 2.5.

As it moves from cells into surrounding body fluids and blood, most of the carbon dioxide reacts with water to form a weak acid (carbonic acid H₂CO₃). This acid ionizes, releasing hydrogen ions (H+) and bicarbonate ions (HCO₃–) which blood carries to the respiratory organs. There, the chemical reactions reverse, and carbon dioxide gas is produced, eventually to be exhaled (see also Chapter 1). In healthy individuals, partial pressure in arterial blood is very close to the partial pressure in expired gases.

So, carbon dioxide production can be considered as a measure of metabolism. In particular, it can be considered a good index for oxidative stress. Indeed, among all the reactions present within cellular respiration there is the oxida-tive phosphorylation that produces not only carbon dioxide, but also reacoxida-tive oxygen species such as superoxide and hydrogen peroxide. This lead to prop-agation of free radicals, which can damage cells and contribute to the onset of many disease.

The increase of CO2 can be due also to physical activity, for example. There is a decrease in case of hypothermia. Also in presence of most forms of lung diseases and some forms of congenital heart disease (for example, cyanotic lesions that result in a bluish-grey discoloration of the skin and in a lack of O2 in the body), there is a decrease of CO2 exhaled.

Also individual’s breathing rate influences the level of CO2 in blood and, as a consequence, in exhaled gas. Breathing that is too slow causes respiratory acidosis (that results in an increase of CO2 partial pressure in blood, which

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Figure 2.5: Glycolysis occurs in the cytosol and does not require oxygen. Aerobic respiration occurs in the mitochondria and only in the presence of oxygen.The products of the reactions include ATP, heat, carbon dioxide and water

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may cause hypertension, build up of heart rate), while breathing that is too rapid leads to hyperventilation, which may cause respiratory alkalosis (that results in decrease of CO2 in blood; so, it can no longer fulfill its role of vasodilator, resulting in arrhythmias, extra systoles).

A way to monitor the carbon dioxide concentration (or partial pressure) is capnography. The max value of capnogram correspond to the end of tidal volume of exhaled breath and the steady-state concentrations of each breath. Carbon dioxide is also one of constituents of tobacco smoke (it is present in 13%).

Ethane (C₂H₆), Pentane (C₅H₁₂), Isoprene (C₅H₈), Butane (C₄H₁₀) and other hydrocarbons

Concentrations of hydrocarbons in human breath have been reported to be in the range of about 0-12 ppb.

Ethane and pentane are saturated hydrocarbons generated from ω6 and ω3 fatty acids perioxidation, respectively.

Lipid peroxidation is a chain reaction which is initiated by the removal of an allylic hydrogen atom through reactive oxygen species (ROS). The radical generated in this way is conjugated, peroxidized by oxygen, and undergoes further reactions(see Fig.Figure 2.6 ).

ω6 and ω3 fatty acids are basic components of cell membranes. In vitro studies have shown that ethane and pentane are generated when cell cultures are exposed to ROS.

Hence, these aliphatic hydrocarbons are regarded, as in vitro and in vivo, markers of lipid peroxidation and, as a consequence, of oxidative stress. Fur-thermore, levels of exhaled pentane and ethane correlate well with other lipid peroxidation markers such as Malondialdehyde (MDA) in blood.

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Figure 2.6: Free radical-mediated lipid peroxidation: possible reactions and reaction products

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Due to the physiological ratio of ω6 and ω3 fatty acids, four times more pentane than ethane is generated through lipid peroxidation. Moreover pen-tane, but not ethane, may easily be metabolized by hepatic cytocrome P450 enzymes . As a consequence, exhaled pentane concentrations should be inter-preted cautiously whenever liver function varies between patients or during the sampling period.

In conclusion, ethane and pentane (as other hydrocarbons, such as Toluene) can be considered good indicators for monitoring the degree of oxidative stress in body.

A further proof comes from a study conducted by Florian Pabst, Wolfram Miekisch, Patricia Fuchs, Sabine Kischkel and Jochen K Schubert ([16]): ox-idative stress mechanism can explain the increased breath pentane in patients with acute myocardial infarction and congestive heart failure.

Not only, as previously mentioned in Fig.Figure 2.1 , breath pentane may in-crease in other conditions, including rheumatoid arthritis, bronchial asthma, schizophrenia, and vitamin E deficiency.

Ethane can be used also as a biomarkers of asthma, vitamin E deficiency, and diseases like scleroderma and cystic fibrosis.

Isoprene is an unsaturated hydrocarbon, and is thought to be formed along the mevalonic pathway of cholesterol synthesis (Fig.Figure 2.7). This is why elevated levels of isoprene in exhaled breath are present in subjects suffering from hypercholesterolemia, or from hyperlipidemia.

Human breath isoprene concentrations show a circadian rhythm with a max-imum at around 6 a.m. and a minmax-imum at about 6 p.m. Concentrations of breath isoprene seem also to be age dependent.

There is experimental evidence that isoprene exhalation may be related to oxidative damage to the fluid lining of the lung and the body. Not only, by

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Figure 2.7: Biochemical pathway of isoprene generation: metabolism of meval-onate

the study mentioned before ([16]) it was demonstrated that exhaled isoprene levels show a correlation with cardiac output: patients having high cardiac index exhale more isoprene than those with low cardiac index.

Higher quantities of isoprene are detected in smokers, too, because of the presence of isoprene in tobacco smoke. Other potential sources of hydrocar-bons there are in the body, such as protein oxidation and colonic bacterial metabolism that generate butane, for example. Tobacco smoke one causes the presence in breath of other hydrocarbons,too, such as toluene, ben-zene, styrene.

Ethanol (C₂H₅OH) and Acetaldehyde (C₂H₄O)

Endogenous ethanol (whose values are very low: 0.62ppm) is originated from microbial fermentation of carbohydrates in the gastro-intestinal tract.

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Figure 2.8: Ethanol breakdown producing acetaldehyde

Then, it enters the blood. As a consequence, it may increase in exhaled gas mixtures because of alcoholic fermentation of an excessive over-load of car-bohydrates.

About the exogenous one, it comes from alcoholic drink (the legal drink-drive limit is typically 1000 times of the typical endogenous ethanol: 153-180ppm). Exogenous ethanol breakdown starts with the partial oxidation of ethanol by enzyme alcohol dehydrogenas, producing acetaldehyde (as shown in Fig.Figure 2.8).

At a later stage, acetaldehyde is converted in acetic acid by acetaldehyde dehydrogenase.

Acetaldehyde is also a significant constituent of tobacco smoke: it is dis-solved into the saliva while smoking. Then, acetaldehyde can be considered a marker for alcoholism, smoking, and also for lung cancer. In fact, ac-etaldehyde is a probable carcinogen in humans, can damage DNA and cause abnormal muscle development as it binds to proteins.

Ethanol metabolism will examine in depth in the following chapter: our in-terest will be focused on this compound, too, because ethanol is one of the

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causes of accumulation of free radicals into the cells, that can result in ox-idative stress, causing arrhythmias and depresses the contractility of cardiac muscle.

Ethylene (C₂H₄)

Ethylene is a biomarker for ultra violet radiation damage of human skin and lipid peroxidation in the lung epithelium.

Methanol (CH₃OH), Hydrogen(H₂), Methane (CH₄)

The origin of breath methanol may be the bacterial fermentation of undi-gested food. Also hydrogen and methane are produced by the breakdown of the carbohydrates in the colon (by the anaerobic bacteria).

As a consequence, they may be correlated to overweight problems, intestinal diseases and probably to an improper life-style.

About the quantities, for hydrogen the baseline value is about 9.1ppm. For methane, its concentration is very low (negligible).

Higher quantities of hydrogen and methane can be found in smokers, because of the presence of methane in tobacco smoke.

Methylated amines (MAs)

Variations in concentrations of methylated amines in human urine, sweat, and breath are associated with various diseases or altered protein metabolism in the body.

Nitrogen monoxide (NO)

In humans, nitric oxide is produced from L-arginine by three enzymes called nitric oxide synthases (NOS): inducible (iNOS), endothelial (eNOS), and

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Figure 2.9: Simplified scheme of the metabolites produced by the interplay between the microbial and human metabolome. After passing through the small intestine,unabsorbed dietary substrates (mainly unabsorbed carbohy-drates and proteins) along with other endogenous substrates (such as bile enzymes, mucus and exfoliated cells) reach the large intestine, where they are fermented by the intestinal microbiota. These microbial metabolites are ex-creted in feces or absorbed and transported to the liver. Subsequently, these absorbed metabolites are subject to further human metabolism and can be excreted in urine and breath.

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neuronal (nNOS). The latter two are constantly active in endothelial cells and neurons respectively, whereas iNOS’ action can be induced in states like inflammation (for example, by cytokines).

In inflammation, several cells use iNOS to produce nitrogen monoxide, in-cluding eosinophils. As such, nitrogen monoxide has been considered an ”inflammometer”.

It was initially thought that exhaled nitrogen monoxide derived mostly from the sinuses, which contain high levels of nitrogen monoxide. It has subse-quently been shown that the lower airways contribute most of the exhaled nitrogen monoxide, and that contamination from the sinuses is minimal. The upper normal level of nitrogen monoxide in different studies ranges from 20 to 30 ppb.

Patients with asthma have higher nitrogen monoxide levels than other people. Their levels also rise together with other clinical and laboratory parameters of asthma (for example, the amount ofeosinophils in their sputum). In con-ditions that trigger inflammation such as upper respiratory tract infections, nitrogen monoxide levels rise.

It has also been noted that factors other than inflammation can increase nitrogen monoxide levels, for example airway acidity and and lung inflam-mation.

Moreover, it is also a vasodilator, so it can be considered a good biomarkers for hypertension.

Oxygen (O₂)

Exhaled oxygen amount is about 13.6%-16%.

Exhaled air has a decreased amount of oxygen and an increased amount of carbon dioxide. These amounts show how much oxygen is retained within

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the body for use by the cells and how much carbon dioxide is produced as a by-product of cellular metabolism.

Lower values may be due to respiration disorders.

Sulfur compounds

An important molecule belonging to sulfur compound is hydrogen sulfide (H₂S).

Its baseline level in human exhaled breath is about 0.33 ppm.

It is usually associated with oral malodour. Breath malodour or halitosis can stem from oral or non-oral sources. There also appear to be several other metabolic conditions involving enzymatic and transport anomalies (such as trimethylaminuria), which lead to systemic production of volatile malodours that manifest themselves as halitosis. Some of the oral causes are periodon-tal disease, gingivitis and plaque coating on the dorsum of the tongue. The non-oral causes of oral malodour include diabetic ketosis, uraemia, gastroin-testinal conditions, irregular bowel movement, hepatic and renal failure and certain types of carcinomas such as leukaemia. Oral malodour may also be exacerbated by a reduction in salivary flow, radiation therapy and some lung conditions including cancer.

Furthermore, hydrogen sulfide is considered also a vascular relaxant agent, and it has a therapeutic effect in various cardiovascular diseases (such as myocardial injury, hypertension).

The body uses sulfur compounds in order to neutralize the action of free radicals: for example, in patients with coronary heart disease, its levels in blood are reduced, as in case of hypertension. On the contrary, in haemor-ragic shock, its plasma levels are increased.

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2.2. HOW TO DETECT VOCS: TECHNIQUES FOR BREATH ANALYSIS

Figure 2.10: Breath sampling system: A) disposable mouthpiece; B) non-return valve; C) Nalophan bag; D) Teflon tube.

2.2 How to detect VOCs: techniques for breath

analysis

2.2.1 Gas chromatography (GC)

Gas chromatography (GC) is a laboratory technique that separates mixtures into individual components. It is used to identify components and to mea-sure their concentrations.

Rather than a physical separation, GC creates a time separation.

First, the gases have to be sampled. The breath sampling system consisted of a disposable mouthpiece, a non-return valve, a Nalophan bag and a Teflon tube (Fig.2.10). The Teflon tube allowed the connection of the bag to the adsorption tube for transferring the breath sample. Each subject is usually asked to calmly fill the disposable bag (approximate volume 3 L) with mul-tiple deep breaths.

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con-Figure 2.11: Sample transfer into the adsorption tube.

densation on the bag walls, then a known volume of sample (250 mL) is flowed through a drying tube filled with 9 g of anhydrous sodium sulphate for water removal and a glass thermal desorption tube pre-packed with 250 mg of 60/80 mesh Tenax GR phase by a pocket pump. The flow is set up on the pocket pump and controlled by a flow meter. During the sample transfer, the sampling bag and the drying tube were kept at 37◦C, whereas the adsorption tube is at ambient temperature (Fig 2.11). Subsequently, the sample is passed through an automated two-stage thermal desorption unit equipped with an internal focusing trap packed with usually 70 mg of Tenax GR.

Then, the gas chromatograph allows the transit of the vaporized mixture (or a gas) through a tube containing a material that retards some components more than others. This separates the components in time. After detection, the result is a chromatogram, where each peak represents a different compo-nent of the original mixture, as shown in Fig.Figure 2.12.

The appearance time can be used to identify each component; the peak size (height or area) is a measure of the amount.

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Figure 2.12: A typical chromatogram.

• A regulated and purified carrier gas source, which moves the sample through the GC. Naturally, the carrier gas must be pure. Contaminants may react with the sample or the column, create spurious peaks, load the detector and raise baselines, and so on.

• An inlet, which works at 200◦_{C, acts as a vaporizer for liquid samples.}

The inlet introduces the vaporized sample into the carrier gas stream. The most common inlets are injection ports and sampling valves. In the first ones, a sort of syringes are used.

• A column, in which the time separation occurs. Because the column type is selected by the user, many different analyses can be performed using the same equipment. Most separations are highly temperature-dependent, so the column is placed in a well-controlled oven.

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Figure 2.13: A GC scheme.

• A detector, which responds to the components as they occur by chang-ing its electrical output. The output from the detector becomes the chromatogram.

• Data interpretation of some sort. The list of times and sizes must be converted to component names and amounts. This is done by com-parison to times and responses of known samples (calibration samples). This can be done manually, but for speed and accuracy, a data system is best.

Some samples are already gases (such as room or outside air, heating gas, etc.) and can be injected directly using either a gas syringe or a gas sampling valve. Most samples are liquids and must be vaporized in order to be ana-lyzed by gas chromatography. This is usually done with a heated injection port in combination with either a liquid syringe or liquid sampling valve. For gas samples, the inlet does not have to vaporize anything so the inlet does not have to be heated.

However, most chromatographers prefer to heat the inlet to ensure that noth-ing condenses in it. A temperature of 100 ˆA◦C is often used.

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Figure 2.14: An example of Gas Chromatograph.

an automatic sampler (equipped with a sample tray and connected to a data system), fully automated analyses become possible.

The separation of a mixture into individual components occurs in the col-umn. Many columns are available to separate mixtures. The choice depends on the nature of the mixture and the kind of information desired. However, all columns function using the same basic mechanism.

How a column separate components?

Let’s see Figure 2.15. It shows a cross-section of a column containing a two-component injected sample (the colored dots). The column is just an empty tube.

If we look again a few seconds later, the appearance has changed. The ”sam-ple” has moved to the right because of the carrier gas flow. It has broadened because of the concentration difference between the sample and the pure car-rier gas surrounding it. The components are still mixed.

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Figure 2.15: The column is just an empty tube

Figure 2.16: Coated column

of the column and repeat the experiment. We can use any coating we wish. In this case, we select one that dissolves the blue-dot component but not the yellow-dot component (Fig.Figure 2.16).

The blue-dot component distributes itself between the coating and the gas. The yellow-dot component stays in the gas phase. When we examine the column a few seconds later, we find that the yellow-dot component is not attracted to the coating.

It moves through the column at the speed of the carrier gas and will emerge first.

The blue-dot component divides its time between the stationary coating and the carrier gas. It travels through the column at a slower speed and will emerge later.

The sample has begun to separate into two peaks.

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• When a vaporized component is presented with a gas phase and a coat-ing phase, it divides between the two phases accordcoat-ing to its relative attraction to the two phases.

• The ”attraction” can be solubility, volatility, polarity, specific chemical interaction, or any other property that differs from one component to another.

• If one phase is stationary (the coating) and the other is moving (the carrier gas), the component will travel at a speed less than that of the moving phase. How much less depends on the strength of the attraction.

• If different components have different ”attractions”, they will separate in time.

A high-resolution column separates peaks down to the baseline. This is much easier if the peaks are narrow (the column is efficient). A small change in flow rate can have an appreciable effect on resolution. Combining the mathematical definitions of efficiency and resolution yields an important result: column resolution is proportional to the square root of column length.

This means that increasing column length is not an effective way to improve resolution. Doubling the column length doubles the analysis time (and the column cost) but only increases resolution by about 40%.

Column selectivity is a less clearly defined property of the stationary phase. Essentially, it is how well a phase differentiates between two compounds. Low selectivity = they elute together. High Selectivity = the peaks separate. How GC detects components?

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There are several detection methods. Let’s see some of them:

• Thermal conductivity (TCD): it operates on thermal conductivity differences. All gases conduct heat, but hydrogen and helium are the best thermal conductors. When either of these is used as the carrier gas, anything else that may be present causes a decrease in the thermal conductivity of the gas stream. This change can be measured and used to create a chromatogram.

• Flame ionization (FID): it responds to anything that creates ions in a flame, which is essentially all organic compounds (there are a few exceptions). In fact, an air/hydrogen flame creates very few ionized particles, but if a carbon-containing material enters the flame, ions production increases. The carrier gas from the column mixes with hydrogen and is burned in air. The FID uses two electrodes, one of which is often the jet where the flame burns, and a polarizing voltage to collect the ions from the flame. When a component appears, the collected current rises. After amplification, the current creates the chromatogram.

• Electron capture (ECD): it has found wide use in environmental work because of its very high sensitivity to halogen-containing compo-nents. A radioactive isotope, usually 63Ni, in the detector cell emits beta particles. These collide with carrier gas to create showers of low-energy free electrons. Two electrodes and a polarizing voltage collect the electrons as a current. Some molecules can capture low-energy electrons to form negative ions. When such a molecule enters the cell, some of the electrons are captured and the collected current decreases.

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After processing, this signal creates the chromatogram.

• Mass Selective (MSD): it identifies components from mass spec-tra; when combined with GC, it the most powerful identification tool available to identify the separated species.

Currently, the Mass Selective method of detection is the most used procedure to identify VOC’s in human breath.

2.2.2 Proton Transfer Mass Spectrometry (PMT-MS)

PMT-MS is another promising field able to analyze analyte concentrations as low as a few ppt. This relies on protonation of the chemical species, coming from a transfer from protonated water.

A PTR-MS instrument (see Fig.Figure 2.17) consists of an ion source that is directly connected to a drift tube and an analyzing system (quadrupole mass analyzer or time-of-flight mass spectrometer).

Almost all VOC’s have proton affinities greater than water, hence there is complete transfer. In fact, the technique is particularly advantageous for breath analysis as large volumetric contributions from N2, O2, CO2 and water do not interfere with measurement. Like MS, species identification is done purely on an m/e ratio.

Commercially available PTR-MS instruments have a response time of about 100 ms and reach a detection limit in the single digit pptv region.

For increased accuracy, PMT-MS is usually coupled with GC to provide additional separation.

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Figure 2.17: An example of PTR-MS instrument.

2.2.3 Electronic nose (e-nose)

Over the last decade, ”electronic sensing” or ”e-sensing” technologies have undergone important developments from a technical and commercial point of view. The expression ”electronic sensing” refers to the capability of repro-ducing human senses using sensor arrays and pattern recognition systems. Since 1982, research has been conducted to develop technologies, commonly referred to as electronic noses, that could detect and recognize odors and flavors.

The stages of the recognition process are similar to human olfaction and are performed for identification, comparison, quantification and other applica-tions.

Generally, e-noses are cheaper, portable and faster, and commonly require less than 30 minutes to analyze a sample.

These devices have undergone much development and are now used to fulfill industrial needs.

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The use of e-noses in medical diagnosis shows promising results in the fields of renal disease, lung cancer and diabetes, even though the devices tend to be too specific and lack cross compatibility, so e-nose is suitable for one par-ticular disease only.

Essentially the instrument consists of head space sampling, sensor array, and pattern recognition modules, to generate signal pattern that are used for characterizing odors.

The detection system, which consists of a sensor set, is the ”reactive” part of the instrument. When in contact with volatile compounds, the sensors re-act(in a reversible way), which means they experience a change of electrical properties.

In most electronic noses, each sensor is sensitive to all volatile molecules but each in their specific way. However, in bio-electronic noses, receptor proteins which respond to specific odor molecules are used. Most electronic noses use sensor arrays that react to volatile compounds on contact: the adsorption of volatile compounds on the sensor surface causes a physical change of the sensor. A specific response is recorded by the electronic interface transform-ing the signal into a digital value. Recorded data are then computed based on statistical models.

Bio-electronic noses use olfactory receptors - proteins cloned from biological organisms, e.g. humans, that bind to specific odor molecules.

The more commonly used sensors for electronic noses are solid state gas sen-sors. The working principle of solid state gas sensors,as mentioned before, is based on the (reversible) interaction of the gas with the surface of a solid-state material, that results in a physical effect (depending on the sensing material), used to achieve the detection of gases in solid state gas sensors. For example, optical processes employ infra-red absorption of gases, whilst

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chemical processes allow to detect the gas by means of a selective chemical reaction with a reagent. The detection of this reaction can be performed by measuring the conductivity change of gas-sensing material, or the change of capacitance, work function, mass, optical characteristics or reaction energy released by the gas/solid interaction.

Organic (as conducting polymers, porphyrins and phtalocyanines) or inor-ganic (as semiconducting metal oxides) materials, deposited in the form of thick or thin films, are used as active layers in such gas sensing devices. The read-out of the measured value is performed via electrodes, diode arrange-ments, transistors, surface wave components, thickness-mode transducers or optical arrangements. Hence, nowadays the number of different solid state based gas sensors is really very large. A brief overview of some of the most important solid state gas sensors is given below.

• Chemo-resistive detectors based upon semiconductor metal oxide films

Popular sensing materials include SnO2, TiO2, ZnO, In2O3, and WO3. Figure 2.20 shows the preparation of an Pt-doped SnO2 chemical sen-sor.

The gas/semiconductor surface interactions, on which is based the gas-sensing mechanism of these sensors, occur at the grain boundaries of the polycrystalline oxide film. They generally include reduction/oxida-tion processes of the semiconductor, adsorpreduction/oxida-tion of the chemical species directly on the semiconductor and/or adsorption by reaction with sur-face states associated with pre-adsorbed ambient oxygen, electronic transfer of delocalized conduction-band electrons to localized surface states and vice versa, catalytic effects and in general complex surface

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Figure 2.18: When the polycrystalline oxide film’s grains are heated (until 400 ˆA◦C),their surface becomes negatively charged and absorbs ambient oxy-gen,giving up to it an amount of free electrons. Because of this reaction,a positive charged region is formed, and works as a potential barrier against the passage of electrons. Indeed, if an electrical current is passed through the sensor, this potential barrier is saw as an electrical resistance (Rs).

chemical reactions between the different adsorbed chemical species (see Figure 2.18 and Figure 2.19).

The effect is a reversible change in conductivity, a feature which is highly sensitive to the composition of the gas.

The changes in the electrical resistance of the sensor are described by the formation of depletion space-charge layers at the surface and around the grains, with upwards bending of the energy bands.

Recent advances in sensor materials has lead to the classification of gases as either oxidizing or reducing, depending on whether the change of sign in conductivity is positive or negative respectively. Metal oxides are classified as p-type if a resistance increase is recorded when inter-acting with a reducing gas, and a resistance decrease when exposed to an oxidizing gas. Logically, n-type metal oxides exhibit the opposite behavior. The classification of n- or p-type semiconductor is used as

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Figure 2.19: In presence of reducing gases, the amount of negatively charged oxygen decreases, resulting in a decrease of the potential barrier and then of the resistance Rs.

it is proposed that this reflects the major type of charge carrier for the two cases, positive charge carrier for the p-type semiconductor, an excess of negatively charged carriers in the case of the n-type.

The gas/solid interactions mentioned can involve changes in the bulk conductance or changes in the surface conductance; as a consequence, two principal types of metal oxide-based gas sensors can be distin-guished. Since the oxygen vacancies are the main bulk defects, changes in bulk conductance are due to changes in oxygen partial pressure at operating high temperatures (600-1000 ˆA◦C), so that the oxygen va-cancies can quickly diffuse from the interior of the grains to the surface and vice versa and the bulk of the oxide has to reach an equilibrium state with ambient oxygen. The main application of this first kind of sensors is the measurement of oxygen partial pressure as required in combustion control systems.

The second principal type of semiconductors gas sensors is based only on changes in surface conductivity at lower temperatures (¡600 ˆA◦C)

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and at quasi-constant oxygen partial pressure. In this condition the sensor detects small concentrations of reactive gases in air by a displace-ment from the constant oxygen pressure equilibrium state, induced by gas interfering effects at the surface of the sensor.

As the working temperature at which these devices work ranges usually from 200 to 400 ˆA◦C, it is necessary to implement a heating element in sensor device.

A simple semiconductor gas sensor is thus basically composed of a sub-strate in alumina or silicon (on which the sensing layer is deposited), the electrodes (to measure the resistance changes of the sensing film) and the heater (commonly a Pt resistive type heater) to reach the op-timum sensing temperature.

A semiconductor gas sensor can work in different operation mode, the most used is based on the monitoring of the sensor resistance at con-stant temperature operation. Other operation modes are the modu-lated operation temperature, the single and/or simultaneous measure-ment of sensor resistance and thermovoltage the single and/or simul-taneous measurement of sensor resistance and temperature.

Vantages in the use of these sensors: low cost, high sensitivity, and a real simplicity in function that allows to combine in the same device the functions of a sensitive element and control electronics.

Disadvantages in the use of these sensors: poor reproducibility, long-time instability due to aging which results in a drift of sensor’s response (long-time instabilities are of considerable importance for the practical use of the sensor; pre-aging thermal treatments and cycle calibration checks have to be carried out in order to avoid the wrong use of the device); non-linear behavior, it means, the change in sensor response

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Figure 2.20: Traditional method in the preparation of a SnO2 sensor.

due to a defined change in gas concentration depends generally on the concentration of the gas to be monitored and also on the concentration of other gases (cross-sensitivity effect); sensor/water vapour interaction leads to an increase in the sensor’s electrical conductance and to a lack of selectivity.

About the cross-sensitivity effect, a different approach to the problem is just based on the development of the electronic nose: it exploits the unavoidable cross-sensitivity of the sensors exactly, instead of trying to eliminate it, and involves a system of pattern recognition which allows to study sensors’ response in a subsequent data processing step.

• Optical gas sensors

They are based on the measurement of changes in absorption spectrum. At present a large variety of optical methods are used in chemical

Development of a new portable device designed for selective olfaction

CORSO DI LAUREA SPECIALISTICA IN INGEGNERIA

BIOMEDICA

Anno accademico 2013/14

Tesi di Laurea Specialistica

Development of a new portable device designed for

selective olfaction

Relatori

Candidata

Ing. Andrea Ginghiali

Danila Germanese

Contents

Introduction

Chapter 1

Something about breathing

1.1

Human respiratory system

1.2

Breathing mechanism

1.3

Respiratory volumes and capacity

1.4

Gas exchanges

Chapter 2

Breath analysis: state of the art

2.1

Molecules and Volatile Organic Compounds

(VOCs) present in human breath

2.2

How to detect VOCs: techniques for breath

analysis

2.2.1

Gas chromatography (GC)

2.2.2

Proton Transfer Mass Spectrometry (PMT-MS)

2.2.3

Electronic nose (e-nose)