Hwi Jin Ko, Eun Hae Oh and Tai Hyun Park
We use the olfactory sense in our daily life for determining the taste of food, as well as the freshness of food, for simply distinguishing malodor from fragrance, and sometimes for perceiving dangerous situations, such as fire. Since a Nobel Prize was awarded to Axel and Buck, who identified the mechanism of olfaction, more research groups have joined the study of an olfactory sensor mimicking olfaction, as well as the olfactory sense itself. Recently, more researches for the artificial sensor mimicking olfaction have been conducted, due to potential applications for diverse areas [1–7]. In particular, the study on a bioelectronic nose using olfactory receptors (OR), which play an important role in olfaction, has a significant meaning, in that human olfaction is expected to be realized in vitro, in addition to the development of an olfactory biosensor. The rapid growth in nanotechnology has also been playing a pivotal role in the progress of the bioelectronic nose. The bioelectronic nose consists of two parts. One is a primary transducer, and the other is a secondary transducer. The primary transducer is a biological part that recognizes odorant molecules and plays a key role in selectivity. The secondary transducer is a nonbiological part, which plays an important role in sensitivity. Primary transducers are biomaterials, such as cells [8], proteins, nanovesicles, or peptides. Various devices, such as surface plasmon resonance (SPR), quartz crystal microbalance (QCM), carbon nanotube-field effect transistor (CNT-FET), carboxylated polypyrrole nanotube-field effect transistor (CPNT-FET), and graphene-based field effect transistor (FET), have been developed as a secondary transducer [3–7, 9–12]. Thus, the performance of the bioelectronic nose has been significantly improved through the integration of biomaterials and nanomaterials; and eventually, bioelectronic noses will have great potential in the applications of a huge variety of fields, such as diagnosis, foods, fragrance and flavor, and the monitoring of environmental pollution.
Usually, the biosensor is a sensing device that detects analytes by the combination of a biological recognition part (primary transducer) with a physicochemical detection part (secondary transducer) (Figure 27.1). Various different biomaterials can be a primary transducer for the biosensor [13]. They are enzymes, antibodies, aptamers, and sensory receptors. Enzymes can be used as a primary transducer, due to the characteristics of their specific binding affinity and biocatalytic activity [14, 15]. Antibodies are efficient biological sensing elements for a biosensor, because of their ability to specifically bind to the target molecules [14]. Aptamers, which are nucleic acids or peptides, bind to specific target molecules and can also be used as a primary transducer for the biosensor [16]. The major role of a biological recognition part is to selectively discriminate target molecules, from a mixed variety of other molecules. Biosensors using sensory receptors as a sensing material have recently made rapid progress. In particular, OR among sensory receptors are useful sensing elements for olfactory biosensors, termed bioelectronic noses. They can effectively discriminate a specific target odor molecule from a large number of target molecules by enhancing the selectivity of the bioelectronic nose.
As mentioned earlier, the biosensor contains a secondary transducer, which is a nonbiological part, in addition to the primary transducer, as a biological part (Figure 27.1). Specific interaction of analytes with biological recognition parts is measured by the sensing element, which produces an electrical signal. The secondary transducer utilizes a nanomaterial-based sensor platform, allowing a biosensor to obtain an extremely high sensitivity. Various nanomaterial-based sensor platforms are able to be utilized as a secondary transducer, such as QCM [10, 11, 17], SPR [18–21], and FET [3, 4, 22]. By combining nanomaterials and biomaterials, a bioelectronic nose can achieve high sensitivity and selectivity.
OR proteins belong to the family of G-protein-coupled receptors (GPCRs) and play a major role in recognizing and discriminating, different molecules [23]. The OR gene family provides the information for the sense of smell [24] and contains about 906 genes, including over 55% of pseudogenes in human, consisting of the largest gene superfamily in the mammalian genome [25–27]. The coding region of all OR genes is approximately 1 kb, without intron [28, 29]. OR proteins are seven-transmembrane proteins, and these structural features are important to their normal functions (Figure 27.2). Human can recognize odorant molecules at the concentration of 10−3 ppb and detect around 10 000 different odor molecules, even though the number of functional OR is about 390. Many research groups have been interested in the development of a biosensor mimicking the human olfactory sense and have tried heterologous expression of OR [10, 11, 30, 31]. Recently, several approaches have tried to develop a bioelectronic nose with high specificity and sensitivity by the combination of OR and a nanomaterial-based sensor platform [1]. The use of OR provides the bioelectronic nose with great improvement in the selectivity.
If we want to make a cell-based bioelectronic nose, it would not be easy. Even though living cells are the best biological sensing materials – by providing an optimal environment for the binding of receptors to ligands and also by mimicking the olfactory signaling system with the closest proximity – problems with respect to stability and reusability still remain. A cell-based sensing system depends on the condition and viability of the cells, and it is difficult to maintain the cell condition and viability at a constant level. In addition, the size of the mammalian cell is too big to apply them to a nanomaterial-based sensor platform, and they generate noise signals when the binding of odorants to the OR is measured through the combination of cells and nanomaterial-based sensor platform, because various metabolic procedures occur in cells, and the senor platform is sensitive enough to generate noise. Therefore, nanovesicles containing an OR and simple signaling component molecules have been developed in order to overcome these limitations of a cell-based sensing system [6, 32, 33].
Since nanovesicles were derived from mammalian cells expressing ionotropic 5-HT3 receptor, and the ligand-specific receptor signaling was successfully measured with a whole-cell-like signaling process [34], nanovesicles have been derived from mammalian cells expressing OR for their application to a bioelectronic nose [6, 32, 33]. The nanovesicles containing OR were constructed by the treatment of mammalian cells expressing OR with cytochalasin B, and the treatment of cytochalasin B makes nanovesicles bud out of mammalian cell membrane. The average diameter of nanovesicles was around 200 nm, with a round shape, and this size is small enough to be immobilized on the nanomaterial-based sensor platform. Even though they are not alive – unlike mammalian cells – the function of nanovesicles can be measured by Ca2+ assay, because they have the components required in olfactory signaling. This characteristic of nanovesicles can exclude noises originating from many metabolic procedures in live cells. Nanovesicles were immobilized on a single-walled carbon nanotube-field effect transistors (SWNT-FET) sensor (Figure 27.3). The binding of odorant molecules to an OR was monitored by OR-mediated signaling on an SWNT-FET sensor platform, with high sensitivity and selectivity. Hexanal, which is one of the indicators of food oxidation, was detected by using nanovesicles containing the OR immobilized on SWNT-FET. This nanovesicle-based bioelectronic nose showed highly selective and sensitive detection of hexanal (Figure 27.4). Overall, nanovesicle-based bioelectronic noses are expected to be widely used for the detection of odor molecules in various industries because they can recognize a specific odor molecule with high sensitivity and selectivity and mimic the signaling pathway in the natural olfactory system; and additionally, they have the advantage of being much more stable than the cell-based system.
OR-based bioelectronic noses have been developed to overcome the limitation of electronic and biomimetic noses. They exhibited high sensitivity and selectivity [3–6, 35]. The OR proteins are seven-transmembrane proteins, and have a regular structure and function, when they are incorporated into a lipid bilayer membrane. If we used the odorant-binding part, which is the peptide derived from the OR protein, it would be much easier to immobilize it on the nanomaterial-based sensor platform. Recently, Lim et al. have developed the bioelectronic nose that uses a peptide receptor derived from an OR protein [36]. The OR-derived peptides were functionalized on SWNT-FET by a single-step process, in which aromatic rings of peptides were immobilized by π–π stacking [36, 37] (Figure 27.5). This peptide receptor-based bioelectronic nose (PRBN) was used to determine the seafood quality in real time by quantitatively measuring trimethylamine (TMA), which is a major odorant molecule that is generated from spoiled seafood.
The PRBN detected TMA at an extremely low concentration of 10 fM and discriminated TMA from other odorant molecules with similar structures (Figure 27.6). Because TMA is an air pollutant and indicator of several diseases, as well as of spoiled seafood, the PRBN specifically detecting TMA has many applications. Much more recently, the bioelectronic nose has been developed in order to detect gaseous TMA for real-time on-site gas analysis [37]. Thus, the use of peptide receptors for the development of a bioelectronic nose is able to overcome the limitation of whole proteins and is expected to provide useful applications in various fields.
The QCM has been used as a secondary transducer for an odorant detection system. Since the QCM, as a piezoelectric crystal sensor, can detect the interaction between small molecules, many researchers used the QCM to detect the odorant by coating the QCM surface with various sensing elements, such as polymer, lipid, enzyme, peptide, and protein. QCM has several advantages, such as simplicity, high sensitivity, and convenience. The principle of QCM is that when specific gas molecules bind to the coating material of QCM, the total mass of the surface increases and this mass change reduces the resonant frequency of the crystal. Due to the ability of QCM to quantitatively detect gas molecules, many researchers have tried to construct an odor sensing system that uses a chemically modified QCM surface. For example, Wyszynski et al. used pegylated lipid, and Koshets et al. used calixarene films as a coating material of the QCM surface to detect various odorous molecules, such as alcohols, esters, acids, and aldehydes [38–40]. However, the chemically modified QCM had difficulty in discriminating structurally similar molecules.
The QCM sensor, functionalized with OR, is a useful odorant sensing system to detect and discriminate various odorants. Several researches show that by combining with an OR, the QCM can be a good odorant sensing device. Wu et al. coated a QCM with OR isolated from bullfrogs in order to construct an odorant sensor [17]. In this study, the QCM coated with bullfrog OR showed a significant change in frequency upon odorant simulation. In another study, the C. elegans OR ODR10 was heterologously expressed in Escherichia coli, and the purified protein was used as a sensing material of the QCM system. When the specific ligand diacetyl was injected in the range of 10−12–10−5 mM, the frequency changed in a ligand-specific and dose-dependent manner [11]. The OR that was expressed in mammalian cell was also used as a sensing material of the QCM system. The HEK-293 mammalian cell expressing the OR I7 was incubated on the surface of quartz crystal. When it was exposed to various odorants, the response was especially high for octanal, which is a known specific odorant of I7; and when it was exposed to various concentrations of octanal, the resonant frequency change increased in a dose-dependent manner [10]. In another study, OR-based synthetic polypeptide was deposited on the QCM to detect a low concentration of acetic acid. In this study, the QCM detected the acetic acid at 10 ppm [41]. These results clearly demonstrate that a piezoelectric system can be a useful odorant detection device, with high selectivity and sensitivity.
The SPR technique is an optical method that uses a surface plasmon wave to monitor the change on the sensor surface. Surface plasmon is the fluctuation in electron density in a solid or liquid caused by incident light. SPR occurs in free electron-like metals, such as silver and gold. The electrons in a metal continuously move freely like a charged cloud. The resonance condition is established when light is focused onto the gold surface. Then, the energy is transferred from the light to the electrons in the surface. The critical angle where the reflectance falls to zero is called the resonance angle. The reflectance is altered by any surface changes or binding events near the surface of the SPR. Thus, the SPR system has been used as a biosensor to detect the interactions of various biomolecules in real time, without labeling [42, 43]. The use of SPR for biosensing was first demonstrated in 1983 [44]. Since then, SPR has been widely used in biological applications, such as ligand screening, cell biology, signal transduction, and DNA–DNA, DNA–protein, protein–protein, and protein–lipid interactions [45–47]. Recently, ligand screening or cellular signaling of GPCR using the SPR system has also been reported [48, 49].
This technique has also been used by Vidic et al. to detect the odorant–OR binding [50]. In this experiment, rat ORI7 was co-expressed with Gαolf in a yeast expression system, and nanosomes containing ORI7 were obtained by mechanical disruption. The nanosomes were immobilized on the surface of SPR, and the desorption of the Gαolf subunit from the lipidic bilayer, through the receptor activation upon the odorant stimulation, was monitored. Similarly, the nanosome containing human OR hOR3A1 was obtained from yeast and used as a sensing material of the SPR system [19]. Cell-based odorant detection using the SPR system was also carried out [20, 21]. The cells expressing ORI7 were cultured on the surface of SPR sensor chips, and the reflectance change was measured upon odorant stimulation. When various concentrations of odorant were injected, the higher concentration of odorant induced a greater change in reflectance. And when various odorants that have similar chemical structure were injected into the cells expressing ORI7, the cells responded specifically to their specific ligand octanal. In this study, it was shown that the SPR system can directly measure the odorant-induced cellular response. Besides, the SPR system was also used to study the functional role of the odorant-binding protein [51, 52].
A nanometer-scale sensor based on nanotube field-effect transistors (NT-FETs) has been developed in the field of chemical sensing. Recently, the SWNT and carboxylated polypyrrole nanotube (CPNT) have been combined with OR to detect the odorant with high sensitivity and selectivity. Kim et al. introduced a SWNT-FET combined with human OR protein as an odorant sensing system [3]. Human OR hOR2AG1, of which the specific odorant is amylbutyrate, was expressed in E. coli, and the partially purified hOR2AG1 was spread onto the SWNT-FET sensor. To detect the odorant binding to OR, the source–drain current was monitored, along with the addition of amylbutyrate. When the various concentrations of amylbutyrate were added, the source–drain current sharply decreased and gradually reached saturation value. This indicates that the binding of odorant to OR induced the change in current. The detection limit was 100 fM, which is significantly lower concentration than the concentration detected using other transducers. This olfactory biosensor also showed the specific detection of odorant when various odorants that have similar chemical structure were added to the sensor. In other experiments, nanovesicle containing OR was used as a sensing material in the CNT-FET sensor system. Jin et al. obtained nanovesicles from mammalian cell expressing hOR2AG1 [32]. The nanovesicles containing hOR2AG1 were immobilized on SWNT-FET, and the activity of the nanovesicles upon ligand stimulation was monitored. The activity of the nanovesicles was generated from the cellular signaling pathway, in which the calcium ions enter into cells upon ligand stimulation. Lim et al. used the nanovesicle-based SWNT-FET sensor as a diagnostic tool to detect the molecules produced from lung cancer patients [53]. A peptide-based SWNT-FET for odorant detection using OR-derived peptide was also reported [36]. Yoon et al. fabricated a FET sensor platform based on hOR2AG1-conjugated carboxylated polypyrrole nanotubes [4]. The hOR2AG1 was immobilized onto a CPNT, and the odorant–OR binding was detected by measuring the change in current. The interaction of odorant and OR can affect the charge carrier density of the conjugated CPNT. The detection limit of this sensor was tens of femtomoles. In 2012, Lee et al. used CPNT-FET to detect the odorant in gaseous phase [35]. Human OR hOR3A1, which was expressed in E. coli, was immobilized onto the CPNT-FET, and exposed to its specific ligand helional in gas phase, to mimic the natural olfactory mechanism. The minimum detectable level of this sensor was 0.02 ppt, which is much more highly sensitive than other gas-phase sensors. These results show that the nanotube based-FET system combined with OR or nanovesicle containing OR or peptide can be used as a highly sensitive odorant detection sensor and can also be applied to various areas that need to detect or diagnose meaningful odorant molecules.
There can be many applications of the bioelectronic nose. Applications include a variety of fields such as medicine, food, fragrance, and environmental monitoring (Table 27.1).
Table 27.1 Applications of the bioelectronic nose
Industry | Application fields |
Medicine | Detection of pathogen |
Early diagnosis of severe diseases | |
Patient condition control | |
Food | Quality control (ingredient confirmation, off-flavor, and spoilage) |
Fermentation process control | |
Detection of bacterial contamination | |
Fragrance and flavor | Consumer's expectation (cosmetics, perfumes) |
Ripeness and freshness of fruits and vegetables | |
Brand recognition (coffee, beer, etc.) | |
Environmental monitoring | Air and water pollution monitoring (malodor, greenhouse gas emission) |
Indoor air monitoring |
Chemical compounds, especially volatile organic compounds from the human body, are important indicators of almost all diseases. These volatile organic compounds are emitted from various parts of the human body, such as the oral cavity, axillae, feet, scalp, skin, and breath; and they provide information about human health conditions, such as infection, diet, stress, immune status, and metabolic diseases [54]. It has been considered since ancient times that volatile organic compounds from the human breath are closely related to human diseases [55], and many researches have demonstrated a variety of key volatile compounds related to diseases. Blood, urine, and sweat are also the sources of odorous compounds that provide a lot of information about human health.
Lim et al. have developed a bioelectronic nose for the diagnosis of lung cancer [53]. Heptanal was a target odor molecule, because it is one of the major odor compounds from patients with lung cancer. The concentration of heptanal is much higher in the blood of patients than non-patients. Human OR, which specifically binds to heptanal, was identified and expressed in mammalian cells. Nanovesicles were derived from mammalian cells expressing the OR and immobilized on single-walled carbon nanotube field-effect transistors (SWNT-FETs) for the development of a bioelectronic nose. The constructed bioelectronic nose detected and discriminated heptanal at a concentration of 100 fM from human plasma, without any pretreatment (Figure 27.7).
A bioelectronic nose using hormone receptors can also be used for medical application because numerous diseases can be induced by hormonal imbalance. Recent study has developed a bioelectronic nose that detects human parathyroid hormone (hPTH) [56]. hPTH receptors were overexpressed in E. coli and were combined with modified close-packed arrays of carboxylated polypyrrole nanoparticles (CPNTs). This hormone receptor-based bioelectronic nose detected hPTH with high sensitivity and selectivity. M1 muscarinic acetylcholine receptors (M1 mAChRs) were also used for detecting acetylcholine (ACh) [57]. ACh is one of the neurotransmitters in the human central nervous system, and dysregulation of ACh in the brain causes numerous neuropsychiatric disorders, such as Alzheimer's disease and Parkinson's disease. The bioelectronic nose device was developed with the combination of overexpressed M1 mAChRs and SWNT-FETs; and it detected ACh at a concentration of 100 pM and selectively differentiated ACh from other neurotransmitters. This bioelectronic nose system using various sensory receptors is expected to be used for the diagnosis of a variety of diseases by using various OR and hormone receptors.
The control and assessment of food quality is an important issue in the food industry, because food quality is closely related to the consumer's choice and the sales volume of the products. Generally, off-odor from spoiled food is generated by the oxidation of food or contamination of food by bacteria and fungi [58–60]. Intake of spoiled foods causes severe health problems. Thus, it is important to discriminate spoiled foods and to select normal and fresh foods.
It is known that the amount of linear aldehydes proportionally increases by the oxidation of unsaturated fatty acids, as foods spoil [61]. Recent study has demonstrated that the bioelectronic nose selectively and sensitively detects hexanal [6]. A bioelectronic nose was fabricated with canine OR as a primary transducer and SWNT-FETs as a secondary transducer and was able to detect hexanal at an extremely low concentration of 1 fM, as well as detected the hexanal in spoiled milk, without any pretreatment. Another approach has assessed seafood quality by measuring TMA from spoiled seafood by the combination of peptide receptor and SWNT-FETs [36]. This sensor was able to determine the degree of spoilage of seafood, without any pretreatment, by measuring the amount of TMA and also to discriminate spoiled seafood, such as oyster, shrimp, and lobster, from other spoiled foods, such as milk, tomato, broccoli, and beef (Figure 27.8). More recently, Ahn et al. have developed olfactory biosensor for detecting fungal contamination in grain [62]. Taken together, the bioelectronic nose is expected to be widely used to assess and control food quality at home as well as in industry.
There has been an increase in environmental pollutants, concomitant with the drastic development of industry, and these pollutants have threatened even our health and ecosystem, as well as the global environment. Environmental pollution is caused by the emission of carbon dioxide (greenhouse gases) and various toxic compounds from industrial processes into the atmosphere, soil, river, ocean, and so on. These pollutions need to be strictly monitored and controlled for the protection of the global environment and of our health from hazardous pollutants. An effective detection system is required to meet these needs.
The bioelectronic nose can be a suitable method for environmental monitoring. One major concern in monitoring environmental pollution is whether OR are still active, even in the dry condition, because air pollution is a severe environmental concern, and also, it is easier to detect the pollutants from the headspace of polluted soil and water samples, without any treatment. A few decades ago, one research group addressed this issue by using a synthetic peptide derived from an OR protein [63]. The sensor using a peptide effectively detected TMA and ammonia, which are well-known air pollutants. Lee et al. also showed that whole OR protein overexpressed in E. coli can detect odor molecules in the dry condition by combining carboxylated polypyrrole nanotubes (CPNTs) [35]. This device selectively detected gaseous odorant at a concentration as low as 0.02 ppt. As mentioned earlier, the OR is expected to have a proper function when incorporated in the lipid bilayer membrane. Recently, bioelectronic nose has been developed for real-time monitoring of water pollution using an SWNT-FET functionalized with olfactory nanovesicles [33]. All these characteristics of the bioelectronic nose show its potential to be used for environmental monitoring.
Potential applications of the bioelectronic nose are wide open in a variety of fields. The quality of our life can be improved through the development of fragrance and flavor industries, and the bioelectronic nose will be used to control the quality of their brands by constantly regulating fragrance and flavor. Process monitoring can be performed using the bioelectronic nose for the production of high-quality products. Another one is the security systems. Unwanted release of confidential documents can be detected by smell sensing. Regarding public safety, currently everybody is threatened with terrorism, for example, by explosives and toxicants. Peoples are concerned about the safety of public places, such as plazas, public buildings, subways, and airports, and drug smuggling is another severe issue threatening public health. Trained dogs are currently used for searching for explosives, toxicants, and drugs. However, an alternative method is needed to replace this method due to many limitations. The high cost of training and maintaining dogs is one limitation, and nose fatigue and the lifetime of dogs as a finder are other limitations. As described earlier, the bioelectronic nose has great potential for a myriad of applications.
The bioelectronic nose was designed using biological materials as a primary transducer and nonbiological materials as a secondary transducer. The primary transducer and secondary transducer play the role of a recognition element and a signal processing element, respectively.
Four types of primary transducers have been developed to enhance the selectivity of the bioelectronic nose. They are cell-, nanovesicle-, protein-, and peptide-based primary transducers. Cell- and nanovesicle-based primary transducers are excellent systems for mimicking the signaling pathway of the human olfactory system and discriminate odorant molecules with humanlike selectivity. However, in the case of cell-based systems, maintenance of the cell is not easy on the chip. In the case of the nanovesicle-based system, the small-sized vesicle is derived from live cells expressing OR and contains all components that are essential for olfactory signal transduction, such as G-proteins, cAMP, and ion-gated channels. The nanovesicle can be easily handled when it is applied to the secondary transducer, due to much smaller size, compared with the cells. However, the nanovesicle-based primary transducer is not reusable. The protein-based primary transducer uses OR proteins, which are produced from a heterologous expression system, such as an E. coli system. OR proteins are more stable, durable, and reusable than the nanovesicles under various measuring conditions. The OR proteins expressed in E. coli expression system may not have a normal tertiary structure. A refolding process is then required. As an alternative approach, peptides derived from OR proteins have been applied to the bioelectronic nose. The peptide-based primary transducer has the merit of high stability and is most suitable for commercialization.
Various kinds of devices have been used as a secondary transducer. The QCM can detect odor molecules with high selectivity and sensitivity by using various coating materials on a quartz crystal surface. However, it makes noise signals very sensitively responding to the environmental disturbance. The SPR can discriminate biomolecules without any labeling in real time, but the equipment for detection is expensive, and miniaturization for on-site measurement is not easy. The carbon nanotube and carboxylated polypyrrole nanotube using a FET extremely enhanced the sensitivity of the bioelectronic nose. The integration of the OR as a primary transducer with the FET sensor platform showed the detection of odor molecules with single-carbon-atomic resolution and femtomolar detection limits.
The bioelectronic nose using an OR was devised with a concept mimicking the human olfactory system, which is well known to recognize over ten thousands of odor molecules, through using only around 390 types of OR. So, the integration of all human OR on a single sensor platform gives the bioelectronic nose a powerful sensing ability, as well as realizing the human nose in vitro. The bioelectronic nose can be widely used for a huge variety of applications, for example, medical application for early diagnosis of severe diseases, food quality control, fragrance and flavor, environmental monitoring for the protection of the global environment and our health from toxic pollutants, and the maintenance of public safety, by blocking threats from terrorism, such as explosives and toxicants. Eventually, human welfare, as well as industrial development, is expected to be much improved through the development and application of the bioelectronic nose.
This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Ministry of Science, ICT and Future Planning (No. 2015065103, 20110031870) and Ministry of Education (No. NRF-2013R1A1A2011174).
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