Exhaled breath analysis of lung cancer patients using metal oxide sensors

CHAPTER 1

INTRODUCTION

Smell used to be a common diagnostic tool in medicine and physicians were trained to use their sense of smell during medical training. Latterly, odour diagnostics have been relegated to secondary status as a diagnostic method. Array based on gas sensor technology now offers the potential of a robust analytical approach to odour measurement for medical use. The technology has been used to examine odours emitted from the body such as from breath, wounds, and body fluids and identify possible problems such as Gastrointestinal, Sinus, Infection, Diabetes, Liver problems. In addition, this technology could be adapted to use inside human body to detect stomach diseases. The exhaled breath analysis to screen lung cancer using metal oxide gas sensors is based on of many clinical inventions on lung cancer detection such as: Metabolomx, a diagnostic company focused on the detection of the metabolomics signature of cancer from exhaled breath, and also inventions of A professor at the Russell Berrie Nanotechnology Institute of the Technion-Israel Institute of Technology, Hossam Haick developed an artificial nose that detects disease biomarkers passing from the bloodstream to the lungs and out through the breath. Breath sampling is completely noninvasive and provides a potentially useful approach to screening lung cancer.Breath analysis can be used as a diagnostic tool because increased or decreased concentrations of some compounds have been associated with various diseases or altered metabolism.

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Exhaled breath analysis of lung cancer patients using metal oxide sensors

1.1 What is in the exhaled breath?

More than 100 compounds have been identified in normal human breath by gas chromatography and mass spectrometry. These volatile organic compounds (VOCs) are produced by metabolic processes and partition from the blood stream via the alveolar pulmonary membrane into the alveolar air. This implies that the concentration measured in breath is related to the concentration in blood. Breath analysis can be used as a diagnostic tool because increased or decreased concentrations of some compounds have been associated with various diseases or altered metabolism. Exhaled breath is largely composed of nitrogen, oxygen, carbon dioxide, water, and inert gases. Trace componentsvolatile substances that are generated in the body or absorbed from the environmentpresent in the nmol/lpmol/l (parts per billion volume-parts per trillion volumes) range make up the rest of the breath. The exogenous volatiles are inhaled into and absorbed through the lungs or absorbed through the skin. They originate from many solvents and petroleum based products. The endogenous volatiles are generated by the cellular biochemical processes of the body. Thus, measurement of VOCs in the breath can provide a window into the biochemical processes of the body.

Fig 1: volatile organic Compounds in exhaled breath fed to sensor array

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Exhaled breath analysis of lung cancer patients using metal oxide sensors

Several classes of VOCs can be measured in the exhaled breath (Table 1). These include saturated and unsaturated hydrocarbons, oxygen-containing, sulfur-containing, and nitrogen-containing compounds. Saturated hydrocarbons (e.g., ethane, pentane, and aldehydes) are formed during lipid peroxidation of fatty acid components of cell membranes, triggered by reactive oxygen species (ROS), these have a low solubility in the blood and hence are excreted in the breath within minutes of their formation.

TABLE 1. Classes of Volatile Organic Compounds in the exhaled Breath

1.2 Exhaled breath of a lung cancer patient

For the pattern of breath VOCs of patients with lung cancer to be unique, the biochemical processes that lead to their generation or metabolism must be different in lung cancer patients than in those without lung cancer.

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Exhaled breath analysis of lung cancer patients using metal oxide sensors The following are a few examples of differences in the biochemical processes of lung cancer cells:

The expression of the antioxidants manganese superoxide dismutase and catalase is different in lung cancer tissue than in normal tissue. This may influence the VOCs identified in the breath.

ROS are increased by cigarette smoke. The VOCs produced as a result of lipid peroxidation from these ROS can be metabolized by cytochrome P450 (CYP) mixed oxidase enzymes. Polycyclic aromatic hydrocarbons in tobacco smoke may induce CYP enzymes.

Easy inducibility seems to be more common in individuals with lung cancer. Adenosine monophosphate-activated protein kinase(AMPK) is a key cellular energy sensor. When cells are faced with energy stresses, such as in tumor microenvironments, AMPK functions to restore energy balance. When active, AMPK functions to inhibit synthetic pathways and stimulate catabolic pathways in an effort to restore levels of adenosine triphosphate.9 Activated AMPK leads to decreased lipogenesis and increased fatty acid oxidation, which can alter the production of VOCs.

The above rationale and support has stimulated investigators to analyze patterns of breath VOCs to determine their potential as a diagnostic test for lung cancer.

TABLE 2: VOCs in exhaled breath of lung cancer patients

CHAPTER 2
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Exhaled breath analysis of lung cancer patients using metal oxide sensors

CONTEMPORARY METHODS FOR SCREENING LUNG CANCER

Lung cancer screening refers to strategies used to identify early lung cancers before they cause symptoms, at a point where they are more likely to be curable. Before screening for any type of cancer can be carried out, doctors must have an accurate and safe test to use. The test must be reliable in picking up cancers that are there. And it must not give false positive results. A false positive result means that a test makes it look as though a cancer could be present when it isnt. For screening to be introduced, we need a test that is simple, quick, not too expensive and not harmful. Lung cancer is often picked up on chest X-ray. But by the time it is diagnosed this way, it is often quite advanced. Researchers are trying to find other screening tests that may help to diagnose lung cancer earlier. At present the following screening methods are used for screening people at high risk of lung cancer: Chest X-ray, Spiral CT scan and Bronchoscopy.

Fig 3: Lung cancer screenings

2.1 Chest X-ray

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Exhaled breath analysis of lung cancer patients using metal oxide sensors Current tests such as X-rays can't usually show early stage cancers and they have some risks. The lungs are very sensitive to radiation and frequent X-rays may cause lung damage. Xrays can also find lung changes that look like cancer and need to be checked by further tests, such as a biopsy, that can cause problems for some people.

Fig 4a: positioning for chest X-ray

Fig 4b: X-ray of chest showing lung cancer

The following are the disadvantages of chest X-ray:

The X-ray may or may not show an abnormality. Types of abnormalities seen in lung cancer include a small nodule or nodules or a large mass. Not all abnormalities observed on a chest X-ray are cancers. For example, some people develop scarring and calcium deposits in their lungs that may look like tumors on a chest X-ray.

The lungs are very sensitive to radiation and frequent X-rays may cause lung damage

2.2 Spiral CT scan

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Exhaled breath analysis of lung cancer patients using metal oxide sensors Spiral computed tomography is a computed tomography technology involving movement in a spiral pattern for the purpose of increasing resolution. Most modern hospitals currently use spiral CT scanners. With CT scanning, numerous x-ray beams and a set of electronic x-ray detectors rotate around the patient, measuring the amount of radiation being absorbed throughout the body. At the same time, the examination table is moving through the scanner, so that the x-ray beam follows a spiral path. A special computer program processes this large volume of data to create two-dimensional cross-sectional images of the body, which are then displayed on a monitor. This technique is called helical or spiral CT.

Fig 5: CT or CAT scan machine with spiral scanning capabilities

Fig 6: (Figure A :) Conventional CT scan. (Figure B :) Spiral CT scan.

If symptoms are severe, the X-ray may be skipped and a CT scan may be performed right away.
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Exhaled breath analysis of lung cancer patients using metal oxide sensors

The advantages of CT scan is that they show much greater detail than X-rays and are able to show the lungs in three dimensions.

Fig 7: CT scan image of lung with small cell lung cancer before and after chemo radiation

The possible disadvantages of helical CT include

The cumulative effects of radiation from multiple CT scans; surgical and medical complications in patients who prove not to have lung cancer but who need additional testing to make that determination;

Risks from additional diagnostic work-up for findings unrelated to potential lung cancer, such as liver or kidney disease.

The screening process itself can generate suspicious findings that turn out not to be cancer in the vast majority of cases, producing significant anxiety and expense.

Some people experience mild itching or hives (small bumps on the skin). Symptoms of a more serious allergic reaction include shortness of breath and swelling of the throat or other parts of the body

2.3 Bronchoscopy

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Exhaled breath analysis of lung cancer patients using metal oxide sensors Bronchoscopy is viewing of the lungs through a lighted, flexible tube (bronchoscope) that is passed through the nose and throat into the main airway of the lungs. The tube, which has a light on the end, allows the doctor to see inside of the lung. If abnormal areas or tumors are seen, cell tissue can be obtained through special tools located at the end of the bronchoscope for evaluation under a microscope.

Fig 8: bronchoscopy
During bronchoscopy, doctor will put a thin flexible tube (bronchoscope) in nose. Then he or she will move it gently down throat to look at the larger airways (bronchi) to lungs. The disadvantages of bronchoscopy are

The procedure is uncomfortable. A local anesthetic is administered to the mouth and throat as well as sedation to make bronchoscopy tolerable. Bronchoscopy has some risks and requires a specialist proficient in performing the procedure. Bronchoscopy is painful.

CHAPTER 3
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Exhaled breath analysis of lung cancer patients using metal oxide sensors

SCREENING OF LUNG CANCER USING METAL OXIDE SENSORS

The system for screening of lung cancer is made by using metal oxide gas sensor array with solid phase micro extraction (SPME) fiber. The system is used to test breath gas of lung cancer patient and health person and results are compared. The screening method using the above technique has four different phases they are: Collection of exhaled breath Characteristics of sensor system Chemical analysis of breath samples Breathing testing with a sensor system

3.1 Collection of exhaled breath

Exhaled breath of lung cancer patients is collected using tedlar bag. The bag size used is 1L and 3L.Exhaled breath sampling is carried out in the morning, before breakfast. After gargling with purified water the gas is collected.

Fig 9: exhaled alveolar breath collection of lung cancer patient Most of the components known to be present in exhaled breath can also be found in the environment. It is therefore necessary to distinguish the breath signal from a contamination with room air. One approach is to simultaneously collect VOCs in the breath and also in the air in order to determine the alveolar gradient (concentration in breath minus concentration in air) of each VOC.

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Exhaled breath analysis of lung cancer patients using metal oxide sensors Second method is to use mouthpiece and aerobic filter are connected for filtering impurities from tedlar bag .Breathing into a machine to deliver a breath sample for early detection of lung cancer is a routine test that is non invasive, safe and simple to do, so likely to be a preventive screening test voluntarily accepted by the patients and will be a mean to detect lung cancer in a very early stage

Fig 10: Mouthpiece and aerobic filter are connected to exhaled breath collector

Fig 11: mouth piece

Fig 12: illustration of exhaled breath collection

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Exhaled breath analysis of lung cancer patients using metal oxide sensors Commonly called Tedlar Bags, gas sample bags are a convenient and accurate means of collecting gas and vapour samples in air. This is particularly true in areas where the concentration is above the detection limits of common analytical instruments. Tedlar bags are manufactured from PVF (Tedlar) film. They are generally considered inert and can be used to collect samples containing common solvents, hydrocarbons, chlorinated solvents, and many other classes of compounds. They are commonly used to collect low-level sulfur gases, but only if the bag fittings are non-metallic (polypropylene, Teflon, or Nylon). Sample hold time will vary for different classes of compounds.

Fig 13: tedlar bags to collect exhaled breath

3.2 Characteristics of sensor system

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Exhaled breath analysis of lung cancer patients using metal oxide sensors Sensor system is made of metal oxide sensor, chamber, data acquisition (DAQ) system, processor, and SPME fiber. Sensor array consist of metal oxide sensor with nanostructure. The SPME fiber is directly exposed to a breath sample to extract and concentrate analyses. The processor is used to carry out number of tasks of data processing using PCA and the data of measuring was saved by personal computer. The system of using SPME fiber shown in Figure14. The data processing can be performed principal component analysis (PCA).

Fig 14: sensor system for exhaled breath analysis

There are two main ways in which the odour can be delivered to the sensor chamber, namely head space sampling and flow injection. In head space sampling the head space of an odorant material is physically removed from a sample vessel and inserted into the sensor chamber using either manual or automated procedure. Alternatively, carrier gas can be used to carry the odorant from sample vessel into the sensor by a method called flow injection. The sensor chamber houses the array of chosen odour sensor; here the sensor chamber houses the Zno nano wire field effect transistor sensor. The sensor electronics connects the output of the sensor array to the data acquisition system as shown in fig 15. DAQ hardware is what usually interfaces between the signal and a Processor. It could be in the form of modules that can be connected to the computer's ports (parallel, serial, USB, etc.) or cards connected to slots in the motherboard.

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Exhaled breath analysis of lung cancer patients using metal oxide sensors

Fig 15: figure depicting the steps in exhaled breath analysis

3.3 Chemical analysis of breath samples

Chemical analysis of breath samples is done to compare the results of exhaled breath analysis by metal oxide sensors with that of gas chromatography and match the results to check the efficiency and accuracy of the former method. VOCs that can serve as biomarker for lung cancer in the breath samples and determined their relative composition, using GC-MS (GC: HP5890, MSD: HP5972) in combination with SPME. Mass range is 29 ~ 55c. The degree of detecting is 28c.

3.4 Breathing testing with sensor system

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Exhaled breath analysis of lung cancer patients using metal oxide sensors The SPME fiber was exposed into tedlar bag of collecting breath and the ambient temperature is 30c. The components of breath gas were adhered to SPME fiber for 30minute. The chamber was clean for 10minute with clean air and the fiber push in chamber with sensor array. The SPME fiber exposed for 5 minute in chamber. The head space sampling method is used here to transfer the odorant material from sample vessel to the sensor chamber; here the sample vessel is nothing but the tedlar bag and the odorant is transferred to the Zno nanowire field effect sensor array i.e. the sensor chamber through the SPME fiber.

Fig 16: Depiction of experimental set up of exhaled breath analysis

3.5 Metal oxide sensor with nano structure

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Exhaled breath analysis of lung cancer patients using metal oxide sensors Sensor array used in this screening technique consist of metal oxide sensor with nanostructure. Zinc oxide nano wire field effect transistor is one of the metal oxide sensors with nanostructure. In the gas sensor according to inventors Rae-Man Park, Sang-Hyeob Kim, Jonghyurk Park and Sunglyul Maeng, a plurality of metal islands are formed on a zinc oxide nanostructure and are independently separated from one another on the zinc oxide nano-structure. The sensitivity to a gas is improved by the metal islands such that a variety of kinds of gases can be detected. The metal islands are formed of one material selected from the group consisting of platinum (Pt), palladium (Pd), nickel (Ni), and cobalt (Co). Diameter = 30-100 nm

Fig 17: Zno nano structure

Fig 18: Zno nano wire with metal islands on it

3.5i Sensing mechanism

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Exhaled breath analysis of lung cancer patients using metal oxide sensors The operating principle of metal-oxide devices is based on the transduction of adsorbed chemicals on the sensor surface with a corresponding change in the electrical conductance. The challenge is to build sensors not only with enhanced sensitivity, but also with the ability to detect specific chemicals in a complex environment (selectivity), and to be quickly reset for the next sensing cycle (refresh ability). With the advent of nanotechnology, nanostructure materials with novel characteristics seem set to address these challenges.

Fig 19: Highly sensitive ZnO nanowire field-effect sensors with electrically controlled sensitivity, refresh ability, and distinguish ability The metal oxide sensing mechanism originates from the charge transfer between the semiconductor and the chemical species adsorbed at the surface oxygen-vacancy sites. The relative change of conductance after exposure to a target gas determines the sensitivity. For example, the conductance of ZnO decreases with the introduction of NO2. At room temperature, with the ZnO in the form of a nanowire, more than 50% of the conductance change was observed under an exposure of 0.6ppm NO2.In comparison, doped ZnO thin films achieved less than 2% conductance change when exposed to 1.5ppm NO2.This demonstrates a high potential for nanowire sensors with superior sensitivity. Moreover, sensitivity can be tuned by exploiting a transverse electric field induced by the gate in the FET configuration. Above a gate threshold (the voltage at which the electrons are depleted in the channel), the sensitivity decreases with increasing gate voltage. This implies that the gate voltage can be used as a knob to adjust the sensitivity range

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Exhaled breath analysis of lung cancer patients using metal oxide sensors

Source Gate

Drain

NW Channel

Fig 20: Chemisorption of VOCS by the Zno nano wire field effect transistor Sensor

3.5ii Refreshing the sensor

The gate potential can electrically desorbs the absorbed gas molecules. Chemical sensing at room temperature is generally not easily reversible because the available thermal energy is usually lower than the activation energy for desorption. This results in a long recovery time. One common method for refreshing sensors is via ultraviolet (UV) illumination, but this increases the complexity of the sensor design, and the recovery time can be fairly long. A gate-refreshing mechanism to improve this situation; as shown in fig 21,

Fig 21. Nanowire sensing response to NO2 and the conductance recovery process induced by a large negative gate voltage pulse.

The conductance of a nanowire can be electrically recovered by applying a negative gate voltage much larger than the threshold voltage. This reduces the chemisorptions rate, and the hole migration to the surface (driven by the negative field) leads to a discharge of
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Exhaled breath analysis of lung cancer patients using metal oxide sensors chemisorbed species. As a result, the channel conductance is effectively returned to its original level. This efficiently refreshes sensors at room temperature without the need for additional hardware.

Fig 22 : Gate refresh voltage magnitude as a function of NO2 (blue curve) and NH3 (red curve) concentrations.

3.6 Solid phase micro extraction fiber (SPME fiber)

SPME is a unique sample analysis technique for complex matrices and for analytes requiring lower levels of detection. SPME eliminates most of the drawbacks associated with extracting organics. SPME requires no solvents or complicated apparatus. Solid phase micro extraction consists of exposing a small amount of extracting phase (coating) associated with a fiber to the sample, for a predetermined amount of time.

Fig 23: parts of SPME device

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Exhaled breath analysis of lung cancer patients using metal oxide sensors

Fig 24: A SPME Device with the Micro fiber Exposed (middle item). Extra needles are available (top item) since the injector syringe can be reused indefinitely. Needles can be reused from 50 to 100 times depending on the composition of the sample. The bottom item is the needle protection guard and GC injection guide.

3.7 Data ACQ board

Data acquisition is the process of sampling signals that measure real world physical conditions and converting the resulting samples into digital numeric values that can be manipulated by a computer. Data acquisition systems (abbreviated with the acronym DAS or DAQ) typically convert analog waveforms into digital values for processing. The components of data acquisition systems include:

Sensors that convert physical parameters to electrical signals. Signal conditioning circuitry to convert sensor signals into a form that can be converted to digital values. Analog-to-digital converters, which convert conditioned sensor signals to digital values.

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Exhaled breath analysis of lung cancer patients using metal oxide sensors Data acquisition begins with the physical phenomenon or physical property to be measured. Examples of this include temperature, light intensity, gas pressure, fluid flow, and force. Regardless of the type of physical property to be measured, the physical state that is to be measured must first be transformed into a unified form that can be sampled by a data acquisition system. The task of performing such transformations falls on devices called sensors

Fig 25: Data ACQ board

Data acquisition applications are controlled by software programs developed using various general purpose programming languages such as BASIC, C, FORTRAN, Java, etc. DAQ hardware is what usually interfaces between the signal and a PC. It could be in the form of modules that can be connected to the computers ports (parallel, serial, USB, Etc.) or cards connected to slots in the motherboard.

CHAPTER 4
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Exhaled breath analysis of lung cancer patients using metal oxide sensors

RESULTS
After SPME injections in the breath sample adsorbed for 30 minute and desorption was performed at GC-injecter for five minutes. In order to avoid the environmental effect, air sample of space to collect was used as a reference. Breath samples of patients with lung cancer and healthy people appeared as table 3. Depending on smoke, the smokers showed benzene toluene, non-smoker was not show. The featured substance of lung cancer patients was appeared decane, tridecane octdecane, heneicosane and undecane diphenylmethane,heptane,3-methyl-hexane etc. These components are similar to previous results of other researchers that are appearing.

TABLE 3: Comparison of patients with lung cancer and healthy exhaled breath S: Smoker, NS: Non-smoker, P: Patient of lung cancer

4.1 Principal component analysis

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Exhaled breath analysis of lung cancer patients using metal oxide sensors

It is a way of identifying patterns in data, and expressing the data in such a way as to highlight their similarities and differences. Since patterns in data can be hard to find in data of high dimension, where the luxury of graphical representation is not available, PCA is a powerful tool for analyzing data. The other main advantage of PCA is that once you have found these patterns in the data, and we can compress the data, i.e. by reducing the number of dimensions, without much loss of information.

Fig 26: PCA of healthy breath and lung cancer breath The results yield well-defined clusters for cancer states and healthy states, thus allowing fast, reliable and noninvasive lung cancer diagnosis. All data points were obtained using the same sensor array.

4.2 software
"Spectra map" is software to create a biplot using principal components analysis, correspondence analysis or spectral map analysis.

In the MATLAB Statistics Toolbox, the functions princomp and wmspca give the principal components, while the function pcares gives the residuals and reconstructed matrix for a low-rank PCA approximation.

CHAPTER 5
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Exhaled breath analysis of lung cancer patients using metal oxide sensors

COMPARISON OF SCREENING METHODS

The following are the disadvantages of chest X-ray:

The X-ray may or may not show an abnormality. Types of abnormalities seen in lung cancer include a small nodule or nodules or a large mass. Not all abnormalities observed on a chest X-ray are cancers. For example, some people develop scarring and calcium deposits in their lungs that may look like tumors on a chest X-ray.

The lungs are very sensitive to radiation and frequent X-rays may cause lung damage

The possible disadvantages of helical CT include

The cumulative effects of radiation from multiple CT scans; surgical and medical complications in patients who prove not to have lung cancer but who need additional testing to make that determination;

Risks from additional diagnostic work-up for findings unrelated to potential lung cancer, such as liver or kidney disease.

The screening process itself can generate suspicious findings that turn out not to be cancer in the vast majority of cases, producing significant anxiety and expense.

Some people experience mild itching or hives (small bumps on the skin). Symptoms of a more serious allergic reaction include shortness of breath and swelling of the throat or other parts of the body

The disadvantages of bronchoscopy are

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Exhaled breath analysis of lung cancer patients using metal oxide sensors

The procedure is uncomfortable. A local anesthetic is administered to the mouth and throat as well as sedation to make bronchoscopy tolerable. Bronchoscopy has some risks and requires a specialist proficient in performing the procedure. Bronchoscopy is painful.

The advantages of screening by exhaled breath analysis

Zinc oxide materials are biocompatible, allowing their use in the body without toxic effects. The flexible polymer substrate used in nanogenerators would allow implanted devices to conform to internal structures in the body. Breathing into a machine to deliver a breath sample for early detection of lung cancer is a routine test that is non invasive, safe and simple to do. Easy to detect lung cancer in a very early stage. There is strong evidence that particulate cancers can also be detected by molecular analysis of exhaled air, even in very early stages of the disease. Breath analysis represents a new non-invasive diagnostic, which can provide information beyond conventional analysis of blood and urine. Exhaled air can be sampled as often as necessary without any restriction. It may even be done for newborn babies, or patients at the intensive care unit.

From the above list of disadvantages of contemporary screening methods and the advantages of the lung cancer screening using exhaled breath analysis it can be concluded that Breath sampling is completely noninvasive and provides a potentially useful approach to screening lung cancer

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Exhaled breath analysis of lung cancer patients using metal oxide sensors

CONCLUSION
The breath tests are attractive since they are noninvasive and can be repeated frequently changing state of critically ill patients. Analysis of exhaled breath for recognition of human diseases using endogenous volatile organic compounds (VOCs) offers the possibility of noninvasive diagnosis. Lung cancer identification by breath test is a rapidly growing area of research that could provide groundbreaking improvements in molecule-oriented screening and monitoring of lung cancer. Breathography is not yet ready to play a similar role in lung cancer screening as that of mammography in screening for breast cancer. However, breath fingerprinting by innovative new means might complement other methods in the early detection of lung cancer.

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Exhaled breath analysis of lung cancer patients using metal oxide sensors

REFERENCES
[1] Joon-Boo Yu, Jeong-Ok lim, Exhaled breath analysis of lung cancer patients using metal oxide sensors First ACIS/JNU International Conference on Computers, Networks, Systems, and Industrial Engineering 2011 IEEE. [2] C. Brambilla, Exhaled biomarkers in lungs- SERIES LUNG CANCER. [3] Roberto F. Machado Departments of Pathobiology and Medical Oncology, Detection of Lung Cancer by Sensor Array Analyses of Exhaled Breath[4] Peter J. Mazzone, Analysis of Volatile Organic Compounds in the Exhaled Breath for the Diagnosis of Lung Cancer. [5] Phillips M, Method for the collection and assay of volatile Organic compounds in breath. [6] David James, Chemical Sensors for Electronic Nose Systems [7] Research and news for Physicians from the Cleveland Clinic Respiratory Institute

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