**2. Basics**

All odor detecting biosensors are based on the fundamental principles of odor sensing in biological system. Therefore, the following section first gives an overview of the biological basics of the olfactory system before addressing the different odor sensor technologies and their classifications. Furthermore, the technical criteria used to create the performance profile are briefly described. At the end of this section, potential markets and applications for odor sensors are discussed.

#### *2.1. Biological Foundations of the Olfactory System*

Before addressing the available odor sensing technologies, the basic principles of odor sensing in biological systems are outlined. The system responsible for the reception of the stimulus initiated by odorants and the transmission and processing of the signals is referred to as the olfactory system. Stimulus perception occurs inside the nasal cavity on a sensory area of a few square centimeters, called the olfactory epithelium. Air reaches the olfactory epithelium via two access points, the orthonasal access during inhalation and the retronasal access during exhalation [23]. Figure 1 illustrates the structure of the olfactory epithelium which mainly comprises three cell types: olfactory sensory neurons, sustentacular cells, and basal cells. The olfactory sensory neurons are primary sensory cells and are constantly renewed as they lie unprotected on the surface and come into direct contact with hazardous substances in the breath [24]. They form dendrites, at the ends of which many small hairs, so-called cilia, are located which serve to enlarge the surface [25]. The cilia protrude into the mucus layer, which protects the skin from drying out and plays an important function in trapping VOCs [26]. The mucus contains water-soluble odorant-binding proteins that form a pocket for binding the VOCs and are therefore considered to play a major role in the transport the VOCs to the olfactory sensory neurons. These neurons contain receptors in the cell membrane of the cilia to which the VOCs bind and thus trigger transduction [23].

The transduction process of sensory olfactory neurons shown in Figure 1 begins upon binding of a VOC to a receptor. This G-protein-coupled receptor is a transmembrane protein in the olfactory cilia of the olfactory sensory cell. Extracellular binding of an odorant results in activation of the receptor, triggering an intracellular signaling cascade that is spatially separated from the receptor. The complex transduction process results in the influx of positive ions, namely calcium and sodium, and the efflux of chlorine ions.

After transduction, the intensity of the stimulus is mirrored in the amplitude of the triggered membrane potential. The potential is conducted to the axon hillock where, after reaching a threshold value, it is converted into electrical impulses of equal amplitude, called action potentials. In this way, the intensity of the stimulus is encoded in the frequency of the action potentials [27].

The process and structure for transmission and preprocessing of the action potentials before they enter the brain is substantially determined by the genetically encoded type of receptors. In the human genome, coding sequences for olfactory receptors are present in approximately 950–1000 genes, although more than 50% of these are nonfunctional due to mutations [25]. Expression of the active genes leads to the formation of a receptor. Only one type of receptor is formed per olfactory sensory neuron [23]. A receptor shows specificity for an odorant or a class of odorants of similar molecular structure. However, activation of the receptor is also possible by other odorants for which the receptor shows a lower affinity. At sufficiently high concentrations, the probability of binding substances for which the receptor shows a lower affinity increases, so that a signal may well result [25]. In addition, the receptor also determines where the signal from the olfactory sensory neuron is directed and processed. Thus, the axons of all olfactory sensory neurons of one

receptor type lead to a collecting point, the so-called glomeruli, in the olfactory bulb. This is a significant first step of signal preprocessing since, therefore, subsequent processing structures only have to process signals from 4000 glomeruli instead of the signals from 100 million olfactory sensory neurons [26]. Accordingly, the principle that one odorant matches one specific receptor type, i.e., one olfactory sensory neuron, whose depolarization leads directly to odor sensation does not apply. Instead, several odorants can bind to several receptor types, i.e., various olfactory sensory neurons, to different degrees, which generates a characteristic pattern of activity in the olfactory epithelium that is transmitted via the olfactory signaling pathway to the brain for pattern analysis [23]. The ability to encode odorants combinatorically results in an almost unlimited differentiability of the very high variability of odorant molecules [25].

**Figure 1.** Structure of the human olfactory system according to Cave et al. [28]. The olfactory epithelium, located in the nasal cavity, mainly consists of basal and sustentacular cells as well as olfactory sensory neurons. On the upper side, neurons run through the cribriform plate of the skull towards the olfactory bulb of the brain. On the lower side, their thin cilia protrude in the mucus layer secreted by the Bowman's gland. Odorant receptor proteins embedded in the ciliary membrane allow the binding of volatile organic compounds (VOCs) solubilized in mucus. Binding of a VOC to the receptor triggers a molecular transduction cascade, as shown on the right, resulting in an influx of mainly calcium ions. Additional efflux of chloride ions is thought to further increase the depolarization.

#### *2.2. Types of Odor Sensing Technologies*

Odor detection technologies can be divided into biosensors and technical sensors, each with several subgroups. Figure 2 illustrates a classification scheme developed in accordance with [20,28–31]. Biosensors contain integrated biological elements, such as cells, cell tissue, proteins, or nanovesicles, which are fundamental for their functionality and rely in part on the structures and processes outlined in the previous section. Technical sensors consist exclusively of technical components and can be divided into so-called electronic noses and conventional instrumental analysis. In the following, these technologies are briefly described.

An electronic nose is a technical system consisting of several chemical sensors that are connected to form a sensor array. Among the various sensor types shown in Figure 2, metal oxide (MOX) and conducting polymer sensors are the most commonly used [32]. These sensors form the detection element of the electronic nose and convert the chemical information into an analytical signal. Depending on the number and type of sensors used, the result of a measurement may be a complex signal pattern [33]. This pattern has to be compared with a reference pattern derived primarily from previous knowledge acquired from an existing data set. Only by matching the signal pattern with the reference pattern, a result with analytical significance is obtained [32]. Conventional analytical methods include, for example, gas chromatography and mass spectrometry. Here, the mixtures of substances in a liquid or gaseous state are examined for their chemical composition

using different physical measuring principles, such as the detection of mass-to-charge ratio in mass spectrometry or polarity in gas chromatography [20]. Biosensors, rather than conventional sensors, use bio-elements such as proteins, nanovesicles, individual cells, or entire cell tissues as recognition elements. In a biosensor, the analyte to be measured docks to a bioreceptor. This creates a specific compound leading to a biochemical reaction that can be technically recorded and evaluated. For example, the reaction may involve a change in the thickness of the bioreceptor layer, the refractive index, light absorption, or electrical charge. These changes are detected by means of a transducer and converted into a signal, which is usually amplified and processed by an electronic system. Thus, a specific signal is generated for each specific substance [34].

**Figure 2.** Overview of different technologies for odor detection. Own illustration, based on [20,28–31].

As depicted in Figure 2, bioelectronic nose technologies comprise of three categories: cell-based, protein-based, and nanovesicle-based. Cave et al. [28] provides a comprehensive overview of these categories, which are briefly summarized below. A number of protein-based approaches aim to utilize olfactory receptor proteins embedded in membrane fragments, nanodiscs, or nanovesicles as the receiving element [35–38]. Membrane fragments of bacterial or yeas<sup>t</sup> cells that express olfactory receptor proteins are solubilized with detergents in order to implement them in the sensor. The detergent acts as solubilizer and mimics the cell membrane environment, so that the olfactory receptor protein maintains its structure and function. This method is limited by missing other proteins, either in cytosol or membrane, relevant for e.g., signal transduction. Moreover, approaches in which odorant receptor proteins are isolated from cell membrane fragments and inserted into nanodiscs, synthetic membranes, including transmembrane proteins that help establishing an environment very similar to the original one, still separate the olfactory receptor proteins. Through sonication and centrifugation or chemical treatment of heterologous cells that express odorant receptor proteins along with proteins responsible for transduction, so-called nanovesicles can be formed. These are small spherical membrane fragments that contain all proteins for complete transduction cascade. Other authors see these approaches as a separate category of bioelectronic noses [29]. They may argue that spherical nanovesicles represent an intermediate form between cells and membrane fragments or nanodiscs. Irrespective of the applied introduced approaches, quartz microbalance electrode, electrochemical impedance spectroscopy, field effect transistors are often used as

detectors. Challenges remain the intensive labor, reproducibility of performance, due to varying amounts of olfactory receptor proteins and variability in performance depending on temperature and humidity. Instead of monitoring the level of interaction of complex odorant receptor proteins with VOCs, simpler peptides derived from odorant receptor proteins can also be used offering the advantage of higher stability. Usually, these peptides are coupled with field-effect transistors and achieve a high sensitivity and reproducibility [39,40]. However, the number of available peptides is currently very limited [28]. As described in Section 2.1, odorant binding proteins are thought to facilitate the transport of VOCs through the mucus to the cilia of the olfactory sensory neuron. Due to this level of interaction with VOCs, they are interesting for an implementation in odorant sensors [41–43]. In comparison to sensors utilizing surface plasmon resonance, electrochemical impedance spectroscopy, sonic acoustic wave resonators, quartz microbalance electrodes those using field-effect transistors achieve a higher sensitivity. Advantages over odorant receptor protein include easier expression in heterologous cells and a higher stability towards environmental conditions. A major disadvantage lies in the limited amount of molecules being recognized due to very low specificity [28,44].

However, protein-based electronic noses have a limited lifetime, as their functionality decreases over time, due to the degradation and denaturation of receptor proteins and peptides. Another disadvantage is that necessary cofactors to restore the initial state after a transduction cascade must be actively provided from the outside, otherwise failure is imminent. Against this background, cell-based bioelectronic noses represent an attractive alternative through their ability to restore defective proteins and to produce the essential cofactors [28]. In approaches focusing on whole tissues, microelectrode sensors are inserted into the olfactory bulb of living animals to measure their neuronal activity when exposed to different odors [45,46]. Analyzing the activity patterns by computer programs to draw conclusions about specific odorants remains a complex challenge. Even approaches that involve existing olfactory epithelial tissue and placing it on microelectrode arrays, for example, encounter similar issues [47]. In addition, the preparation effort is very high and the comparability is low, since different signals are obtained depending on the recording area on the epithelium. The use of olfactory receptor organs from insects allow an easier analysis of distinct activity patterns, but the organs show stability issues [48,49]. Instead of using whole tissues, approaches using dissociated olfactory sensory neurons measure only single neurons that are either suspended, immobilized on a microelectrode array, or trapped in microfluidic chambers. The proof-of-concepts available so far, encounter the problem that there is no method to isolate or organize neurons of a specific receptor type, resulting in the readout signal being arbitrary [28,50–52].

Considering the advantages of cell-based sensor methods over peptide or proteinbased ones as well as the outlined drawbacks of strategies utilizing whole tissues and dissociated olfactory sensory neurons, approaches using cultured cells are considered one of the most promising methods [11,28]. In cultured cells approaches, specific DNA segments encoding olfactory receptor proteins from olfactory neurons are transfected into a heterologous cell line so that it expresses olfactory receptors. Alternatively, there are strategies to extract olfactory receptor proteins and implant them into heterologous cells. The cells require a suitable culture environment (nutrient medium) on the sensor platform to remain functional and viable [28]. Veithen et al. [53] present a number of cell lines that are typically considered for in vitro expression and implantation of olfactory receptor proteins initially, regardless of the selection technology used. These include heterologous mammalian cells, such as the human embryonic kidney 293 cell line (HEK293) and Hela cells, as well as insect cells, Xenopus oocytes and yeas<sup>t</sup> cultures [54–60]. To enhance the delivery of the expressed olfactory receptor molecule into the cell membrane, additional DNA segments of receptor transport proteins (RTP) are transcribed so that the cell line stably expresses chaperones such as the RTP1, RTP1s, RTP2, in addition to the expression of the olfactory receptor protein [56]. If the display of olfactory receptor proteins on the membrane succeeds, the binding of a VOC to this receptor triggers a reaction process

that is comparable to the transduction cascade explained in Section 2.1 and also leads to the influx of calcium ions. One method to visualize this influx is by calcium imaging based functional assays. Calcium-sensitive indicators are used to detect deviations in the intracellular calcium concentration. For example, GCaMP is a genetically encoded calcium indicator that changes structure when calcium ions bind to it, activating its attached green fluorescent protein (GFP). Other commonly used calcium-sensitive dyes include Fura-2 and Fluo-4 [53,61]. Cell-based approaches are not limited to optical readout techniques, for instance, the surface plasmon resonance method or microelectrode arrays can also serve as transducers [28].

#### *2.3. Technical Performance Criteria of Odor Sensing Technologies*

In order to describe the performance of an odor sensor, both static parameters, such as selectivity and sensitivity, and dynamic parameters, such as service life, can be considered. In the following, the individual criteria considered in this paper are described. The selection and definitions were developed in the course of an expert workshop of the authors based on Fraden et al. and verified by the review and supplementation of external experts in different fields of sensor technology [62].

Measurement quality:


Handling:


Technical construction and production:


#### *2.4. Markets and Application Fields for Biosensors*

The annual turnover of all suppliers in the biosensor market was USD \$11.5 billion in 2014 and is expected to grow to USD \$28.78 billion by 2021. This corresponds to a growth rate of 12.2% per year [63]. In the following, the fields of application for the use of odor sensors are listed and described regarding their market volumes in Germany. Figure 3 gives an overview of the findings.

**Figure 3.** Overview of market volumes in billion euro (EUR) of different application fields in Germany [64–69].


correct composition of the products, in order to be able to analyze odors and develop them more specifically, for example [20].

