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The field of organic-borne biomarkers has been gaining relevance due to its suitability for diagnosing pathologies and health conditions in a rapid, accurate, non-invasive, painless and low-cost way. Due to the lack of analytical techniques with features capable of analysing such a complex matrix as the human breath, the academic community has focused on developing electronic noses based on arrays of gas sensors. These sensors are assembled considering the excitability, sensitivity and sensing capacities of a specific nanocomposite, graphene. In this way, graphene-based sensors can be employed for a vast range of applications that vary from environmental to medical applications.
- graphene
- graphene-based sensors
- biomarkers
1. Introduction
1.1. Biomarkers
The medical field is constantly seeking newer technologies and procedures that can enable successful, accurate and rapid diagnostics. The current diagnostic methodologies often rely on interventions that are invasive, painful, time-consuming and, at times, even unsafe to the patient, not to mention the commonly elevated costs and the lack of repeatability of the results [1,2]. To remedy these limitations, newer options have been studied regarding their suitability to diagnose pathologies and health conditions. Among those options, the detection, identification and quantification of biomarkers is gaining relevance [2,3].
A biomarker can be defined as any biological-borne molecule or organic feature, like the temperature, whose characteristics or detection can be indicative of the presence or development of a vast range of health conditions and pathologies [4]. In fact, the identification of biomarkers is often used to assess eventual health risks, to screen health state, to determine prognostics, to evaluate the response to treatments and even to evaluate the progress of a disease [5,6].
The most often studied biomarkers are compounds that are produced by the human organism as an outcome of the development or evolution of a specific pathology or health condition [7,8]. These molecules, once produced, interact with the biological tissues and are commonly released to the exterior of the organism through several body fluids. Breath, urine, faeces, perspiration, blood and even lacrimal fluid are examples of fluids that often carry molecules whose characteristics can make them act as biomarkers for diseases [9,10,11].
Among all the potential molecules studied as potential biomarkers, some of them can be linked to a vast range of conditions while others are studied regarding their exclusive relationship with a single disease. Acetone, for example, has been deeply addressed in the medical community since it can be used as a biomarker for at least 11 pathologies, namely sleep apnoea [12], malaria [13], lung cancer [14], gastric cancer [15], diabetes [16], cystic fibrosis [17], COVID-19 [18], colorectal cancer [19], chronic liver disease [20], chronic kidney disease (CKD) [21], and asthma [22]. Coincidently, the detection of isoprene in human emissions can equally be linked to 11 health conditions, which are chronic obstructive pulmonary disease (COPD) [23], chronic kidney disease [24], chronic liver disease [25], breast cancer [26], cystic fibrosis [27], COVID-19 [28], gastric cancer [29], diabetes [30], malaria [31], lung cancer [32], and sleep apnoea [12]. On the other hand, compounds like 2-acetylpyridine (chronic obstructive pulmonary disease [33]), ethanal (COVID-19 [28]), ethyl acrylate (cystic fibrosis [34]), 2-, 3- and 4-ethyltoluene (squamous cell cancer [35]), 5-ethyl-2-methylheptane (tuberculosis [36]), methyl butyrate (prostate cancer [37]) and many others are relatable with a single disease.
1.2. Graphene-Based Sensors
Graphene-based sensors have been largely explored regarding their vast range of applications, their adaptability to distinct scenarios and their proven results in the identification and quantification of specific molecules. Nonetheless, this exploration is only possible due to the characteristics of graphene whose excitability and sensitivity allow its application in the development of molecular electronic devices for sensing purposes.
Initially discovered by Geim et al. (2007) after peeling off the thin (atomic size) layers from pieces of graphite, graphene and graphene-derivative materials have played an important role in sensing-based scientific fields [46]. In fact, graphene is often described, at an atomic level, as the lightest, thinnest and strongest material, which presents hydrophobic behaviour, is aggregable in aqueous solutions, insoluble in organic solvents, and can be stable at temperatures as high as 200 °C [47]. Regarding its structure, graphene presents symmetric arrangements of bonds between carbon, which form a honeycomb pattern with a surface area of around 2600 m2/g [48].
Besides the mentioned features of graphene, it is worth saying that its major advantage is related to the fact that graphene characteristics can be easily altered if exposed to several scenarios. In fact, graphene can be chemically altered through π–π interactions or via electron transfer processes, for example, when exposed to scenarios rich in organic compounds whose functional groups are effortlessly attachable to graphene. In addition, graphene is an electrical conductor whose optical and electrical properties can be modified in several ways that include chemical, electrochemical and thermal options [47,49]. This reactivity and excitability of the graphene and graphene derivates materials make them ideal to be employed in several types of sensors, namely, optical fibre sensors, physical sensors, chemical sensors, electrochemical sensors, and wearable sensors [48,49].
Evidently, each one of these types of sensors can be assembled through different techniques. One of the preparation methodologies of graphene-based sensors often used to analyse gaseous or volatile samples is the layer-by-layer technique. Here, alternated electrically charged thin films of polyelectrolytes are applied to a solid base containing interdigitated electrodes [50]. Examples of those polyelectrolytes are polyethyleneimine and, evidently, graphene oxide. The application consists of successive immersions of the base in the solutions
2. Graphene Sensors for Biomarker Detection
2.1. Asthma and COPD
Asthma, chronic obstructive pulmonary disease and other health conditions that involve inflammatory processes of the respiratory tract are among the most common and rather mortal pathologies worldwide. In fact, the World Health Organization (WHO) has reported a staggering number of 500,000 deaths directly caused by asthma, and 3 million deaths provoked by COPD worldwide every year [60,61]. These kinds of pathologies impose a very poor quality of life on the patients, whose state depends directly on the rapidness and effectiveness of the treatments [62,63]. In order to prescribe the proper treatment procedures, the physicians have to accurately diagnose the pathology in question; however, the current methodologies to do so are often time-consuming, expensive, invasive and very discomforting to the patients [64,65].
The detection, identification and quantification of volatile organic compounds in the breath of patients has gained relevance as a rapid, accurate, non-invasive, painless and low-cost procedure to identify inflammatory pathologies of the respiratory tract. Compounds such as acetone [22], decane [66], propanol [67], hexane [67], dodecane [66], ethylbenzene [68] and many others have been linked to the diagnosis of asthma when detected in exhaled air. A similar logic can be applied to the diagnostic of COPD via the identification of acetaldehyde [33], benzaldehyde [23], benzene [23], butanal [33], isoprene [23], isopropanol [33], limonene [69], nonanal [70], and many other analytes in breath.
Besides the potentialities of the biomarkers in breath, one of the current challenges to be surmounted is the lack of standardized procedures for the detection of those analytes. The field of graphene oxide-based sensors has positioned itself as one of the potential candidates to help overcome this issue. In fact, several works have been developed in the area [45,71].
The field of graphene oxide-based sensors has been making its contribution to the development of systems for the analysis of breath samples. The work of Kumar et al. (2020) is an example of that. The authors have developed an array of sensors based on graphene oxide composite to study three specific volatile compounds, ammonia, ethanol and the potential asthma biomarker, acetone. Standard samples of acetone were previously prepared with concentration levels of 1000 and 2000 ppmv. The developed sensors were capable of detecting, identifying and quantifying acetone successfully, proving their suitability for the eventual analysis of breath samples and asthma diagnosis [74].
Another well-known asthma biomarker is propanol [67]. This volatile organic compound has been studied through sensors of graphene oxide in several scientific studies. One of those works was developed by Samadi et al. (2021). The authors developed sensors capable of detecting propanol at room temperature by scattering thin layers of a specific nanocomposite, ZnO@SiO2/rGO. Then, the sensor was tested by being exposed to standard samples of propanol previously prepared with concentration levels ranging from 150 ppmv to 450 ppmv. Propanol was successfully identified and quantified, proving the suitability of this kind of system for the assessment of biomarkers in breath [75].
In order to detect the mentioned compounds in breath, some work has been performed regarding the graphene oxide sensing capacity. Murashima et al. (2016), for example, addressed the capacities of graphene-based sensors for the qualification and quantification of one of the aforementioned biomarkers, i.e., acetaldehyde. To do so, the authors developed the sensors by scattering thin films of graphene oxide that were later exposed to gaseous samples of acetaldehyde. The samples were prepared in 10 to 50% ratios between the target analyte and room air. All the samples were successfully detected, proving that the sensors developed by the authors can be utilized for clinical purposes [77].
2.2. Chronic Kidney Diseases
A fast and accurate diagnostic is especially mandatory in the case of chronic kidney diseases. This group of pathologies is responsible for leading thousands of people to hospitals every year due to direct complications of the pathology but also due to all the secondary consequences and comorbidities [81]. When identified in later stages of development CKD often results in mandatory haemodialysis treatment, acute renal failures and even life-threatening cardiovascular episodes [82,83].
Considering the aforementioned facts, it is crucial that newer, faster and more accurate procedures to diagnose CKD are developed. Electronic noses, whose working principle is based on the sensing capacities of graphene-based sensors, have been developed to tackle this challenge by qualifying and quantifying the analytes emitted in the breath of the patients that can act as biomarkers of CKD [84]. It is worth stating that several scientific studies have been published regarding this issue and whose focuses are given to known CKD biomarkers, namely, ammonia [85], acetone [86], ethanol [87], and isoprene [24], among others.
Aiming to assess the suitability of graphene-based sensors for the detection of volatile organic compounds often detected in breath, Lee et al. (2021) developed and validated reduced graphene oxide-based sensors and machine learning algorithms. The system was then tested with three specific volatile organic compounds, isopropanol, acetone and ammonia. As mentioned, ammonia is a common biomarker of chronic kidney diseases in the exhaled air so, with that in mind, authors exposed the sensors to several standard samples of the analyte, at specific concentration levels, and were able to identify it with accuracy levels above 90%. They were equally capable of differentiating health volunteers from chronic kidney disease patients, gathered in a synthetic cohort of volunteers, with accuracy levels above 20% [88].
Two specific volatile compounds commonly related to the diagnosis of chronic kidney diseases are acetone and ethanol. With that in mind, Tung et al. (2020) developed graphene-based chemoresistive sensors that were later used to detect specific volatile organic compounds, including acetone and ethanol. Other analytes equally relevant to the field of carcinogenic biomarkers were also tested; they are methanol, chloroform, acetonitrile and tetrahydrofuran. The developed array of sensors could detect the target compounds in limits of detection as low as 2.82 ppbv, outstanding results that undoubtedly show the suitability of graphene-based systems for lung cancer diagnosis via biomarkers in breath [90].
Acetone was also the target compound of the study developed by Choi et al. (2014). As stated by the authors, human exhaled air has tremendous advantages and a high potential to act as a valuable source of information about the organism and about specific pathologies and health conditions. In this way, and being aware of the relationship between the presence of acetone in breath and an eventual diagnosis of conditions like diabetes or gastric cancer, among others, the authors developed an array of sensors using WO3 hemitubes functionalized by graphene-based electronic sensitizers. To test the sensors’ performance, solutions of acetone were prepared with 1 ppmv concentration. The sensors were then exposed to the solution and their response was assessed. A full detection of the target VOC was achieved with limits of detection as low as 100 ppbv and with response times ranging between 11.5 and 13.5 s. The outstanding sensitivity and overall behaviour of the sensors leave no doubts about their suitability for real medical scenarios [91].
Ammonia is a well-known volatile organic compound that has been studied regarding its direct connection to haemodialysis treatments. This compound occurs naturally in the human organism since it is produced by protein metabolism and is often excreted through urine. Nonetheless, it can traverse biological tissue and is often emitted through breath after being transported to the lungs via the circulatory system [94].
Aiming to identify ammonia in the exhaled air of patients undertaking haemodialysis treatment, Shahmoradi et al. (2021) fabricated graphene-based sensors, namely, sulfonate graphene-, graphene oxide- and reduced graphene oxide-based sensors. Then, the authors exposed the produced sensors to previously prepared gaseous samples of ammonia whose concentrations ranged from 0.5 ppbv to 12 ppmv, similar concentration levels to the ones commonly found in the exhaled breath of haemodialysis patients. The system proved to be suitable for an accurate and sensitive detection of the target analyte in a non-invasive and painless way. The results prove the auspicious future of graphene-based sensors in the field of detection of biomarkers for the evaluation of the organism’s reaction to haemodialysis treatments and to diagnostic renal disease overall [95].
2.3. Diabetes
Diabetes is among the most common pathologies worldwide, with notable incidence in developed countries. The WHO estimates that the number of patients suffering from diabetes will reach the humongous number of 350 million cases by the year 2030. Coincidentally, a considerable portion of the cases are diagnosed in a later stage of development, preventing a proper treatment and leading to relevant and, in some cases, life-threatening comorbidities [97,98].
The current procedures for monitoring blood glucose, besides being effective, are often invasive, complicated, and even expensive, so medical academia is constantly seeking new and non-invasive procedures that allow us to control diabetes accurately and rapidly [99]. In order to tackle this limitation, the scientific and medical communities have given full attention to the detection and identification of volatile organic compounds emitted in the breath that can act as biomarkers for the diagnosis of diabetes [100]. The main diabetes biomarkers are acetone [101], methanol [102], ethanol [102], isoprene [30], isopropanol [101] and others.
In order to help the medical field detect and identify the compounds emitted in breath, the scientific community investigating graphene-based sensors has placed their efforts in developing novel sensors, devices and even methodologies that allow a full characterization of the emitted analytes [103].
A direct example of the utility of graphene-based sensors can be found in the work of Kalidoss et al. (2019). In one of the first papers on the matter, authors developed gas sensors based on a ternary (graphene oxide, tin dioxide and titanium dioxide) nanocomposite for the detection of acetone in the breath of diabetic patients. Then, the authors exposed the developed sensors to several concentration levels of the analyte and fully characterized the behaviours of the sensors, namely, their response and recovery times, their ideal operating temperature, and even their sensitivity. The results achieved by the authors prove the promising future of this type of sensor [104].
During a second work, the authors developed and tested graphene-based chemoresistive sensors with the single purpose of detecting acetone in exhaled air samples. To do so, a prototypic device was developed around the array of sensors and used to analyse breath samples of 17 diabetic patients and 13 healthy volunteers. The authors claim that their system allows the differentiation among both groups of volunteers with an accuracy of over 60%, proving the suitability of graphene-base sensors for this type of application.
Acetone was also the biomarker of diabetes targeted by Thakur et al. (2022). An array of six sensors comprising hybridized graphene oxide field-effect transistors was developed by the authors specifically for the detection of acetone. To do so, the array was exposed to a dummy breath, i.e., a synthetically prepared breath whose purpose was to mimic the human exhaled air. The samples were prepared with synthetic air and specific portions of acetone whose concentration levels ranged between 400 ppbv and 80 ppmv.
In a direct application of graphene oxide nanosheets, Choi et al. (2014) focused their work on the sensing capacities of graphene oxide and developed gas sensors to detect the presence of acetone in the exhaled air of diabetic patients. Samples of acetone, previously prepared with concentration levels ranging between 1 and 5 ppmv, were used as target materials. The sensors were exposed to the samples and their behaviour was analysed. The authors state that the developed sensors enabled the detection of acetone with high levels of selectivity and limits of detection as low as 100 ppbv. This value proves the suitability of the described procedure to perform analyses of exhaled air, since the concentration of acetone in breath is often superior to 100 ppbv [108].
2.4. Gastric Cancer
The diagnostic of gastric cancer often involves a gastric endoscopy with a complementary biopsy and subsequent identification through histopathological analysis. As is known, this type of procedure is extremely invasive and leads the patient to scenarios of extreme discomfort. Additionally, the results often require some time to be available causing a delay in the treatment procedures. In this way, the development of non-invasive, rapid and accurate techniques for gastric cancer diagnosis is required to allow proper treatment of the disease as rapidly as possible [112,113].
Aiming to accelerate the diagnosis of the pathology, the identification of gastric cancer biomarkers in breath has gained pertinency [114]. One can find several works whose scopes were dedicated to the identification of biomarkers; they are acetic acid [115], acetone [116], 2-butanone [117], isoprene [115], propanal [115], phenyl acetate [116], furfural [118], toluene [119], and many others.
To overcome the challenges of detection and analysis of volatile organic compounds, the field of electronic noses based on graphene oxide sensors has played an important role. A considerable number of works have been published regarding the capacity of graphene for the sensing of breath biomarkers [120].
Acetic acid was one of the analytes targeted by Moura et al. (2023) in their recent study on volatile compounds. The authors developed graphene oxide sensors based on thin films scattering for the purpose of sensing four specific analytes, methanol, isopropanol, ethanol and acetic acid. Then, the authors exposed the sensors to samples of acetic acid ranging from 24 to 120 ppmv and successfully detected and quantified all the samples. The entire procedure and, specifically, the resolution of 0.04 ppmv achieved by the sensors prove their eventual applicability to analyse exhaled air samples [50].
The diagnostic of gastric cancer, as mentioned in the initial portion of this chapter, often requires invasive and painful procedures. The field of graphene-based sensors has made a contribution to overcoming these issues. The detection of biomarkers in the breath of gastric cancer patients, in this way, has grown as a potential solution. Once overcome limitations like the lack of standardized procedures, the lack of portability of the developed systems and the full identification of the target analytes, graphene-based sensors can be, without doubt, a useful tool for the medicine of the future.
2.5. Lung Cancer
Due to its direct connection to the respiratory system, lung carcinoma has been deeply studied regarding the possibility of a faster, more accurate, non-invasive and painless diagnostic that would allow a proper and effective treatment in expedited time. This is, in fact, one of the main necessities of current medicine, accurately and rapidly diagnosing lung cancer [122,123]. The high levels of incidence allied to the mortality of the pathology make it one of the most concerning conditions worldwide. In fact, every year, more than 2 million new cases are diagnosed on the planet [124].
Several approaches, procedures and methodologies have been used to detect lung cancer. The detection of biomarkers in the exhaled air is one of them [125,126]. One can list several analytes often linked to pulmonary carcinogenic conditions; they are heptane [127], hexanal [128], pentane [32], 2-butanone [129], furan [130], decane [14], acetone [32], isoprene [131], ethanol [132], or even formaldehyde [133], among many others. To supplant the issues of analysing and identifying the volatile analytes emitted in breath, the field of graphene oxide-based sensors has given its contribution by developing innovative electronic noses based on the sensing capacities of graphene [134]. In fact, the literature provides some examples of that contribution.
Graphene-based sensors were also the basis of the analytical technique employed by Shanmugasundaram et al. (2022) to study two specific compounds, decane and heptane. These compounds are well-known biomarkers often detected in the breath of lung cancer patients [6]. To detect them, the authors developed a methodology based on SnO2 nanospheres and a reduced-graphene-oxide-incorporated SnO2 nanocomposite. Then, the sensors were exposed to standard samples of the target gases, allowing the authors to fully assess the behaviour of the developed system during the exposure. The sensors proved to be capable of detecting concentration levels as low as 1 ppmv and the authors intend to test them in real samples of breath [136].
A final biomarker often linked to the diagnosis of lung cancer is formaldehyde, as mentioned. In order to assess its real suitability for helping to diagnose lung cancer in a rapid, painless, noninvasive and accurate way, Shanmugasundaram et al. (2022) developed an electronic nose whose operating principle was based on reduced graphene oxide superstructures. The authors simulated exhaled air samples of healthy people and lung cancer patients by preparing solutions of formaldehyde with specific concentration levels, namely, 49 ppbv to healthy samples and 83 ppbv to pathological samples. Once exposed to the cohort, the developed sensors were capable of fully differentiating between both groups with pinpoint accuracy. This outstanding evidence proves the suitability of graphene oxide-based systems to diagnose lung cancer through biomarkers in breath [140].
2.6. Sleep Apnoea
It is estimated that a staggering number of 936 million adults suffer from sleep apnoea worldwide [141]. This pathology is characterized by profound alterations in the breathing rhythm, i.e., variations between breathing and non-breathing periods during sleep due to the collapse of the airways. The patients are, then, exposed to a vast range of commodities and consequences from this successive oxygenation interruption [142,143].
As with other respiratory pathological conditions, sleep apnoea has been studied regarding eventual biomarkers emitted in the breath that can lead to a rapid, accurate and non-invasive diagnosis. The main analytes often linked to sleep apnoea are acetone [12], decanal [12], heptane [12], hexane [12], nonane [12], octane [12], β-pinene [144], toluene [12], and p-xylene [12], among some others. Due to the current challenges in the detection of these analytes in samples of breath, several procedures have been developed around an array of sensors based on the sensing capacities of a specific nanocomposite, graphene [145].
Once again, acetone has shown itself as a common compound commonly present in the exhaled air and often linked to health conditions. Sleep apnoea is no exception cit. With that in mind, Sen et al. (2021) developed an array of sensors based on ZnO-SnO2 nanocomposites decorated with reduced graphene oxide. Then, the authors tested the sensors regarding their sensing capacities by exposing them to solutions of acetone prepared at concentration levels ranging between 1 and 10 ppmv. The outstanding accuracy of 91% and detection limits of 0.675 ppmv prove not only the perfect capacity for volatile acetone detection but also the suitability for the identification of biomarkers in breath [147].
Isopropanol, also known as isopropyl alcohol, is another biomarker of sleep apnoea whose detection has been studied with graphene-based sensors. An example of that is the work of Ray et al. (2022). Aware that the breathomics is the future of non-invasive, painless, accurate and rapid medicine, the authors developed graphene-based sensors that were then used to analyse real breath samples. The breath samples were collected and spiked with concentrations of 0.5 ppmv of well-known volatile organic compounds: acetone, acetaldehyde, butanediol, cyclohexanone, decane, ethanol, methanol, octane, styrene, propyl benzene and, as previously addressed, the sleep apnoea biomarker, isopropanol. Outstanding levels of accuracy (ranging between 92.8 and 96%). Considering the achieved results, there is no doubt about the total suitability of graphene-based sensors for the identification of biomarkers in breath [150].
This entry is adapted from the peer-reviewed paper 10.3390/s23229271
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