Recent Advances and Applications of Odor Biosensors

Abstract

Many odorants fall outside the sensing scope of the human olfactory system, yet they play quite important roles in our daily lives. Thus, numerous devices have been invented for qualitative or quantitative odor detection issues. Some analytical instruments, e.g., gas chromatography–mass spectrometry, are precise and reliable, but also expensive and bulky. Odor sensors with a smaller size and a lower cost play an important role in on-site rapid odor detection. The sensitivity and selectivity of these sensors are mainly determined by their sensing materials. Inspired by the powerful animal olfactory system, researchers extract diverse biological materials and combine them with transducers to form odor biosensors. In this paper, we introduce odor biosensors based on transducer types such as microelectrodes, fluorescence, surface plasmon resonance, field-effect transistor, quartz crystal micro-balance, etc. Then, we list several applications of odor biosensors, such as environmental monitoring, disease diagnosis, food quality control, and security. In addition, we analyze the future development of odor biosensors.

1. Introduction

Some researchers insist that humans can discriminate more than 1 trillion olfactory stimuli [1]. Despite having such a powerful and sophisticated olfactory system [2,3,4,5,6,7], there are still plenty of odorants that cannot be directly detected by the human sense of smell owing to sensitivity, safety, and detection range limitations [8,9,10]. To solve this problem, various devices have been proposed, including, for example, gas chromatography combined with mass spectrometry (GC-MS) [11,12,13], Raman spectroscopy [14,15,16], ion mobility spectrometry (IMS) [17,18], odor sensors [19,20,21], and so on. Among them, GC-MS, IMS, and Raman spectroscopy devices are bulky and require precise operation, while odor sensors with a small size, low power consumption, and a low cost are widely used in different scenarios [22,23]. The development of gas sensors could be traced back to the 1950s, when J. Eaton et al. measured minute air contaminants based on the surface tension change in a liquid drop in 1954 [24]. In 1961, R.W. Moncrieff designed an instrument for measuring and classifying odors and the film-coated thermistor was fixed on the Wheatstone bridge, wherein the odor carried by air induced a current signal [25]. A modern odor sensor system was invented in 1982 by Persaud and Dodd who used a metal oxide sensor array to mimic the discrimination of the mammalian olfactory system at a gross level [26]. Then, the commercially available electronic nose formed by conducting a polymer sensor array [27] and the electronic tongue for liquid-phase odor detection [28] were introduced, followed by more and more high-performance odor sensors being developed.

Odor sensors are classified based on their sensing mechanisms, i.e., transducer types, as follows: quartz crystal microbalance (QCM), surface plasmon resonance (SPR), field-effect transistor (FET), surface acoustic wave (SAW), microelectrode, and fluorescence [29,30]. These transducers themselves are not sensitive or selective towards odorants. Therefore, sensing materials are essential for capturing the target molecules, and utilizing proper sensing materials is key to enhancing sensor performance. Enlightened by the superior capability of the animal olfactory system, multiple biological materials—for instance, the aptamer, odorant-binding protein (OBP), olfactory receptor (OR) protein, olfactory sensory neuron (OSN), and olfactory epithelium—are integrated with transducers to create odor biosensors [31,32]. These biosensors inherit the characteristics of animal olfaction and can convert target odor information into a readable signal (Figure 1), thus being widely utilized in manifold aspects such as environmental monitoring, disease diagnosis, food quality control, and security [33]. On the other hand, some issues also exist in odor biosensors, e.g., low reproducibility, short lifespan, complex operation, and so on. Thus, many efforts have been made by researchers for better odor biosensors [34].

Several odor biosensors review papers have been published before. These papers mainly focused on biological sensing materials [35,36,37,38]. This review consists of three parts. First, we present some typical odor biosensors according to their sensing mechanisms and list corresponding instances. Then, we enumerate current application fields of odor biosensors. Finally, we illustrate the future perspectives of odor biosensors.

2. Odor Biosensors with Different Transducers

2.1. Microelectrode-Based Odor Biosensors

The microelectrode method measures the current [39], potential [40], or impedance variation [41] when the interaction between ligand molecules and the sensing material happens [42,43]. Thence, biological materials with spontaneous potential are suitable for current and potential measurements. Microelectrodes are fabricated via microfabrication techniques, either as planar arrays on a flat insulating substrate [44,45,46,47,48] or as needle-shaped probes [49,50] with microfabrication technology. When placed on a plane, microelectrodes always form an array, named a microelectrode array chip [51,52,53]. Multiple microelectrodes increase the data density and are capable of odor discrimination. The sensing materials, e.g., isolated OSN or olfactory epithelium, come into contact with the microelectrodes for signal recording (Figure 2a,b). As for needle-shaped microelectrodes, they could be inserted into a biological organism [54], cell complex [55], or single cell [39] (Figure 2c–e). Both invasive measurement and in vitro olfactory epithelium signal recording [56] shorten the lifetime of a biosensor. With the advancement of microfabrication technology, researchers have attempted to create an in vivo biosensor that could monitor real-time odorant-induced response for one week or even longer working periods [57,58,59]. This technique has also promoted the study of animal olfaction, including, for example, the quantitative evaluation of odor masking [60].

Measuring impedance spectroscopy variations could allow one to detect the combination of odorant molecules and sensing material. The measurement part consists of a counter electrode, a working electrode, and a reference electrode [61]. The sensing material could be an OR protein [62,63] or OBP [64,65], and recently, OBP has been preferred due to easy protein expression and purification. To obtain the desired OBP, a DNA sequence encoding the OBP is expressed in the host cell. After harvesting the host cells, the supernatant is handled for the OBP material [65]. The procedure for obtaining the OR protein is more complex because OR is embedded in the cell membrane. The OR gene is cloned into a vector, then transformed into a host cell. After culturing them for a sufficient amount of time, the cells are lysed, and the sample is centrifuged. The protein in the supernatant is purified with a His-Trap column and a Vivapsin column [63]. The immobilization of the protein on the bare electrode is not very stable; thus, N-(3-dimehylaminopropyl)-N′-ethylcarbodiimide hydrochlori (EDC) and N-hydrox-ysuccinimide (NHS) are introduced for achieving dense steady protein coverage [66,67,68] (Figure 2f). The expression of the protein is then confirmed by Western blotting [69,70,71]. The density of protein coverage on the transducer surface can be confirmed by atomic force microscopy (AFM) or X-ray photo electron spectroscopy (XPS) measurements [72]. The normalized impedance change is obtained by calculating the electron transfer resistance for evaluating the biosensor’s properties.

Figure 2. Microelectrode-based odor biosensors: (a) isolated OSN on a microelectrode chip; (b) olfactory epithelium stripped from rat nasal cavity; (c) needle-shaped microelectrode inserted into the olfactory bulb; (d) needle-shaped microelectrode inserted into a cell complex; (e) needle-shaped microelectrode inserted into a single cell; and (f) microelectrode covered with OBP for measuring the impedance spectroscopy. Regarding the above, (a) is reprinted from [73] with permission from Elsevier, (b) is reprinted from [44] with permission from Elsevier, (c) is reprinted from [54] with permission from Elsevier, (e) is reprinted from [55] with permission from John Wiley and Sons, and (f) is reprinted from [66].

Figure 2. Microelectrode-based odor biosensors: (a) isolated OSN on a microelectrode chip; (b) olfactory epithelium stripped from rat nasal cavity; (c) needle-shaped microelectrode inserted into the olfactory bulb; (d) needle-shaped microelectrode inserted into a cell complex; (e) needle-shaped microelectrode inserted into a single cell; and (f) microelectrode covered with OBP for measuring the impedance spectroscopy. Regarding the above, (a) is reprinted from [73] with permission from Elsevier, (b) is reprinted from [44] with permission from Elsevier, (c) is reprinted from [54] with permission from Elsevier, (e) is reprinted from [55] with permission from John Wiley and Sons, and (f) is reprinted from [66].

2.2. Fluorescence-Based Odor Biosensors

In the fluorescent method [74,75,76], cells with fluorescent dye [77] or fluorescent protein [78,79] are employed as the sensing materials. Once the target ligand is captured by the OR on the cell membrane, an ion channel opens and then the ion influx emerges. The increase in intracellular ion concentration enhances the emission light intensity and is regarded as the biosensor response. To obtain the cell with fluorescent dye, OSN is preferred because it already has expressed OR protein and an ion channel on its membrane. The isolated OSNs are incubated with calcium-sensitive fluorescent dye (e.g., Fluo-4 AM or Fluo-8 AM) [80,81] for around 20 min and are then ready for subsequent experiments (Figure 3a). These OSNs usually form a sensing matrix for more robust biosensors [77,82]. The OR types in one animal vary from several hundred to several thousand [83,84,85], which means that the OSN matrix has a good latent detection capability. However, OSN also has several drawbacks: First, the proportion of OSNs in the olfactory epithelium is around 4–9% [77,82] and most cells trapped in the sensing matrix have no relationship to odor detection. Second, the OR type of OSN is unclear, and a standard odorant sample or single-cell reverse transcription polymerase chain reaction (RT-PCR) is necessary for confirming the expressed OR on OSN [82]. Third, the fluorescent dye is not always stable, and the OSN cannot be passaged for many generations.

On the other hand, researchers have focused on cells with fluorescent proteins in recent years [86,87,88,89]. In some of these studies, cells that could passage for multiple generations (for instance, Sf21, Hana3A, or HEK293) [87,90,91] were selected and expression vectors containing OR and fluorescent protein (usually sensitive to calcium ion or cyclic adenosine monophosphate) [29,87] genes were constructed [92]. Then, cells were transfected with expression vectors and cultured for around 1 week to establish a stable cell line (Figure 3b). The expression level of OR and fluorescent proteins was checked with PCR or Western blot methods. The OR type was determined by the operator. Hence, when a specific target ligand needed to be gauged, researchers could find and express its corresponding OR. After the cell line was established, the sensing material’s preparation in the following experiments was low-cost and simple. Meanwhile, the nanovesicle that secreted from the cell line also contained the same proteins. It could be exploited in fluorescent biosensors [93]. In addition, the fluorescent protein was also used to label a specific OSN for in vivo biosensors [58].

2.3. SPR-Based Odor Biosensors

Surface plasmon resonance is an optical phenomenon; its sensing mechanism comprises molecular interactions near the surface of nanoparticles shifting the SPR spectra peak [94]. When the size of the nanoparticle which forms the surface of the sensor is equal to or smaller than the wavelength of incident light, free electrons collectively oscillate, named localized surface plasmon polaritons [95,96,97,98]. This collective oscillation has its maximum absorbance at the resonant wavelength. Scientists can quantify the surface binding through monitoring the changes in resonance wavelength [99,100,101,102].

The sensing materials used in SPR-based biosensors are cells [100], membrane fragments [103], nanosomes [104], OBP [102], liposomes [105], and so on (Figure 4). The conformation changes in these sensing materials are captured by SPR-based biosensors. As cells per se can adhere to a gold surface, no additional substance is required [106]. OBP or OR proteins usually require EDC+NHS [103] or an antibody [105] to assist with the immobilization procedure. OR proteins need to be inserted into the membrane or liposome to maintain their seven transmembrane structures for normal function. On the contrary, OBP is directly secreted into the mucus for carrying hydrophobic odorant molecules. So, it is expressed, purified easily, and kept bioactive in vitro. Besides odorant detection, SPR-based odor biosensors can gauge the affinity of OBP with OR proteins [107]. This helps researchers gain a deeper understanding of the role of OBP in animal olfaction.

2.4. FET-Based Odor Biosensors

The FET sensor has gate, source, and drain electrodes. When the gate voltage is above the threshold voltage, carriers move in the channel between the source and drain electrodes, and the FET sensor can be used for odor detection when its threshold voltage changes [108,109,110,111]. Bare FETs have no selectivity towards odorants. Hence, their modification by sensing materials is necessary. The sensing mechanisms of FET-based biosensors is the modulation of drain source current.

In most FET biosensor application conditions, electrolyte or insulating layers cover the drain and source electrodes [112], and the gate electrode (usually Ag/AgCl electrode) is submerged in a buffer solution. To fabricate the desired structure, researchers first created a carbon nanotube [113,114,115,116,117] or graphene [72,118,119] pattern. In carbon nanotube FET (CNTFET), a photoresist is patterned on a silicon oxide wafer via standard photolithography [117]. Then, the patterned wafer is submerged in a CNT solution for CNT adhesion. Finally, the metal electrodes are formed by photolithography, thermal evaporation, and the lift-off process (Figure 5a). When it comes to the graphene FET, a single graphene layer is loaded on the silicon wafer using chemical vapor deposition and dry-transfer methods [120]. The graphene pattern is fabricated on a silicon oxide wafer using photolithography and reactive ion etching processes. Finally, the drain and source electrodes are constructed using the same methods in CNTFET (Figure 5b). Nanoparticles are also suitable for loading the sensing protein (Figure 5c) [121]. Proteins [116], peptides [72], and aptamers [122] are commonly used in FET-based biosensors for odor detection owing to their small size and high sensitivity. With the extended gate, cells can also be used for FET-based odor biosensors (Figure 5e) [123].

2.5. QCM-Based Odor Biosensors

QCM is a kind of resonant sensor based on the piezoelectric effect of quartz crystal [124,125,126], which almost linearly transforms the mass change absorbed on the QCM surface into its resonant frequency change [127]. There are many quartz cutting types—for example, AT cut, BT cut, and SC cut—depending on the quartz cutting angles [128,129,130,131]. Among them, the AT cut is the most used due to its good frequency–temperature characteristics. A typical QCM sensor has a sandwich structure, where the middle layer is piezoelectric quartz crystal, and its upper and lower surfaces are coated with thin (~100 nm) metal electrodes. Once an alternating current is applied on the electrodes, the quartz crystal undergoes a reciprocating motion. When the alternating electric field frequency matches the quartz crystal intrinsic frequency, resonance occurs, and the mechanical deformation and vibration intensity reach their zenith. In the resonant state, the substance attached to the electrodes results in the resonant frequency change, thereby enabling the sensing ability.

In the very beginning, QCM-based odor biosensors used the cell membrane fraction for odor detection [70,71,132]. HEK293 or E. coli was transfected with a plasmid containing the OR gene and then expressed OR protein on its plasma membrane. The membrane fraction was directly spread on the electrode or immobilized with the assistance of an aptamer (Figure 6a). Considering that the lipid in the membrane had no contribution towards odor detection, scientists extracted the OR protein or OBP as the sensing materials (Figure 6b) [133,134]. In order to further improve the effectiveness of QCM-based odor biosensors, a peptide mimicking OBP was introduced [135]. Researchers attempted different amino sequences in the peptide for more advanced sensing capabilities.

2.6. SAW-Based Odor Biosensors

QCM and SAW are both acoustic sensing methods. Their difference is the acoustic mode type: QCM uses bulk acoustic waves while SAW uses surface acoustic waves. Interdigital electrodes are fabricated on the piezoelectric substrate (e.g., LiNbO₃, ZnO, AlN, SiO₂) [136,137]. Once an alternating electrical signal is applied to the transmitter electrode, an acoustic wave is generated. This acoustic wave propagates along the interface of the substrate [138]. When it reaches the receiver electrode, the electrode’s characteristic size (for example, width) is an integer multiple of the acoustic wave’s half wavelength. The acoustic wave resonates in the interdigital channel and is converted into an electrical signal via the direct piezoelectric effect [139]. The gaps between the transmitter and receiver electrodes are covered with a sensing layer, where the target analyte is adsorbed. The mass loading of target molecules leads to variations in frequency, phase, or acoustic velocity, which are considered the sensor response [140,141]. The wave types of SAW devices are listed as follows: first, the Rayleigh wave (R-SAW), mainly used for gas-phase detection; second, the shear horizontal wave (SH-SAW), suitable for liquid-phase detection; and third, the Love wave (LW-SAW), similar to shear horizontal wave with slower speed and less attenuation [142]. By comparing their characteristics, it is not difficult to see that Love wave-type SAW sensors have the widest application prospects [143,144].

Similarly to QCM-based odor biosensors, the sensing materials in SAW-based odor biosensors are mainly antibodies [145], OR proteins [146], and OBP [147,148] (Figure 7). The sensing layer, which is thinner than that in QCM-based sensors, is preferable due to its attenuation. These proteins are first suspended in buffer and then added to SAW transducers for odor detection.

The comparison of different transducers in odor biosensors is shown in

Table 1. Comparison of different transducers in odor biosensors.

Transducer Type	Detection Principle	Sensing Material	Response Time	Feature	References
Microelectrode	Current, potential, and impedance change	OSN, OB, and olfactory epithelium	Around ten seconds	Multiple cells or sites; invasive condition with high sensitivity but short lifetime; non-invasive condition with easy operation but low sensitivity	[39,73]
Fluorescence	Fluorescent intensity change	OSN and cell expressing OR	Around half minute	Multiple cells; non-invasive with long lifetime; easily affected by ambient light	[82,87,88]
SPR	Resonance wavelength change	Cell, OR protein, and OBP	Several seconds	Rapid and label-free detection; good stability and reproducibility; complex operation	[103]
FET	Conductivity change	OR protein, OBP, and aptamer	Several seconds	Very high sensitivity and selectivity; complex fabrication	[72,116,119]
QCM	Resonant frequency or dissipation change	OR protein and OBP	Several seconds	Low cost; high sensitivity; complex experimental setup	[127,134]
SAW	Resonant frequency change	OR protein and OBP	Several seconds	High sensitivity; moderate reproducibility	[140,141,143,144]

. The selection of transducers should consider specific measurement requirements.

2.7. Other Odor Biosensors

Besides the aforementioned biosensors, some other biosensors can also be used for odor detection.

LAPS has electrolyte, insulator, and semiconductor layers; its surface is flat and thus suitable for sensitive material loading [149]. When a laser beam irradiates the LAPS semiconductor layer, electron hole pairs are generated. Meanwhile, the bias voltage generates a depletion layer between the semiconductor and the insulation layer. When the electron hole pairs excited by light diffuse into the depletion layer, due to the electrical properties of the electrons and holes themselves, they are torn apart by the electric field between the semiconductor and insulating layer and move towards the two poles to form a current. At this point, if a modulated light source with a certain frequency irradiates the semiconductor layer, a phenomenon of alternating movement of electron hole pairs occurs, allowing for the detection of alternating photocurrent in the external circuit [150]. The interaction between the sensing layer and the target substance changes the original membrane potential balance of the sensitive layer, and with the continuous change in the concentration of the target substance to be measured, the membrane potential also changes accordingly, thus achieving the purpose of quantitative detection of the target substance [151]. In past studies, olfactory mucosa tissue was isolated from rat nasal cavity and the cilia receptor side was placed upwards on the LAPS [152,153]. Odorant-induced extracellular potential changes could be recorded by the LAPS. The OSN itself or OSN heterologously expressing OR could also be used for odor detection [154,155,156,157] (Figure 8a). To extend the sensing material lifetime and maintaining stable characteristics, HEK293 cells were utilized to replace OSN [158].

In another study, OR and Orco were inserted into a bilayer lipid membrane to imitate a cell [159]. When the OR captured its target ligand, the ion flow emerged, resulting in the current variation (Figure 8b). The Ag/AgCl electrodes at the bottom recorded the ionic current as a biosensor response [160]. The ion gate formed by OR/Orco could be substituted by a nanopore-forming membrane protein (e.g., alpha-hemolysin) [161]. In this condition, the aptamer carried the odorant molecules to trigger the current spike.

3. Odor Biosensor Application Fields

3.1. Environmental Monitoring

There are numerous volatile organic compounds (VOCs) in the ambient environment. Some are produced in nature while others are artificial odorants. These odorants are related to environmental pollution conditions and the state of survival and reproduction of animals and plants [162]. Hence, it is necessary to detect them precisely.

Actinomycetes’ and blue-green algae’s metabolism produces geosmin [163]. Geosmin has an earthy smell and is regarded as a contaminant in tap water [119,164] (Figure 9a). Termtanasombat et al. selected the Drosophila olfactory receptor Or56a, which is quite sensitive and selective to geosmin [165]. In that paper, the sensing cells that expressed diverse ORs only responded to their corresponding stimulus. In order to extend the biosensor detection range from the liquid phase into the gas phase, Deng et al. kept a thin liquid layer on the cells which allowed the target ligand to penetrate the buffer and maintained cell viability [166].

The substances produced by human activities are a major source of environmental pollution. Fujii et al. combined aptamers and nanopores together for sensing omethoate, a commonly used pesticide [161]. Agarose gel and lipid were alternately added into two similar small chambers, and then the nanopore protein and DNA aptamers were applied to the agarose gel. When the omethoate vapor was introduced, the aptamer captured the pesticide molecules and passed through the nanopore, resulting in a current spike (Figure 9b). The intensity of the current spike had a positive correlation with the vapor exposure time and showed no significant difference when other stimuli were applied, thereby proving the effectiveness of this biosensor.

Odor source localization was an important auxiliary technology for environmental monitoring [167,168]. Terutsuki et al. mounted silkmoth antennae on a portable electroantennogram and used it as the odor sniffer in a small drone [169]. The electroantennogram signal intensity was determined by the odor concentration, thereby guiding the drone to fly towards the odor source (Figure 9c). Although the odor source localized in this paper was pheromone bombykol, insect antennae can be modified through bioengineering to be sensitive to other odorants for achieving different odor source localization.

Figure 9. Odor biosensors applied in environmental monitoring: (a) OR embedded in nanovesicles for geosmin and 2-methylisoborneol detection, * p < 0.05, ** p < 0.01, *** p < 0.001 in the one-way ANOVA model; (b) DNA aptamer capturing an omethoate molecule and then passed it to the nanopore to trigger a long current spike, DNA aptamer before (i), during (ii), and after (iii) translocation through the nanopore; and (c) antenna from male silkmoth combined with a drone for odor source localization. (a) is reprinted from [164] with permission from Elsevier. (b) is reprinted from [161] with permission from the Royal Society of Chemistry. (c) is reprinted from [169].

Figure 9. Odor biosensors applied in environmental monitoring: (a) OR embedded in nanovesicles for geosmin and 2-methylisoborneol detection, * p < 0.05, ** p < 0.01, *** p < 0.001 in the one-way ANOVA model; (b) DNA aptamer capturing an omethoate molecule and then passed it to the nanopore to trigger a long current spike, DNA aptamer before (i), during (ii), and after (iii) translocation through the nanopore; and (c) antenna from male silkmoth combined with a drone for odor source localization. (a) is reprinted from [164] with permission from Elsevier. (b) is reprinted from [161] with permission from the Royal Society of Chemistry. (c) is reprinted from [169].

3.2. Disease Diagnosis

The smells from human sweat [170], urine [23,171,172,173], and breath [174,175,176,177,178] indicate an individual’s health condition. The accurate analysis of these odors allows one to identify disease signs, reduces the pain of traditional invasive inspections, especially for chronic disease monitoring, cancer screening, or early warning of infectious diseases, provides dynamic data support for personalized diagnosis and treatment, reduces medical costs, and improves public health prevention and control efficiency.

Cho et al. detected 2-ethyl-1-hexanol from headspace gas in lung cancer and decided to use it as a cancer biomarker [93] (Figure 10a). They checked human OR libraries and found that human OR 4D11P was sensitive to this biomarker. To obtain long-term stable sensing capability, they heterologously expressed the OR on HEK293 cells and generated nanovesicles containing OR 4D11P. Finally, they confirmed the functionality of this cell line via 2-ethyl-1-hexanol stimulation and a calcium signaling assay. Other odorant molecules could also be lung cancer biomarkers.

To fully utilize the sensing ability of OSNs, Suzuki et al. fabricated a microchamber array and filled it with isolated OSNs [82]. The OSNs that responded to biomarkers were retrieved from the microchamber array and then subjected to single-cell RT-PCR (Figure 10b). The ORs from responsive OSNs were expressed on HEK293 cells and retained the same detection capabilities. Hu et al. developed a biosensor consisting of peptides and MXene [179]. This biosensor could detect fifteen odor molecules affiliated with five categories of alcohols, ketones, aldehydes, esters, and acids by pattern recognition algorithms (Figure 10c). Furthermore, the biosensor sensed the breath samples from healthy populations and patients with lung cancer, upper digestive tract cancer, and lower digestive tract cancer. With machine learning assistance, the detection accuracy for all patients was higher than 90%. This cost-effective and precise model is suitable for non-invasive tumor diagnosis.

Besides cancer-related biomarkers, Lee et al. invented a QCM-based biosensor for low concentrations of ammonia in human breath [180], as ammonia has a close relationship with the digestive system and kidney problems (Figure 10d). Acetone in the breath has a smell of rotten apples, which is closely related to diabetic ketoacidosis. In reference [175], an acetone bio-sniffer was developed to measure the exhaled breath acetone concentration and assess lipid metabolism. A fiber-optic bio-sniffer was manufactured by attaching a flow-chamber with nicotinamide adenine dinucleotide (NADH)-dependent secondary alcohol dehydrogenase (S-ADH)-immobilized film onto a fiber-optic NADH measurement system (Figure 10e). The excitation light source was a diode emitting 335 nm ultraviolet light. NADH was consumed by the enzymatic reaction of S-ADH, and the consumption was proportional to the acetone concentration. We believe that these biosensors will be widely used in the field of rapid, non-invasive, and low-cost disease detection in the near future.

3.3. Food Safety

Food safety is directly related to public health, social stability, and economic development. Unsafe food causes foodborne illnesses, ranging from mild abdominal pain and vomiting to organ damage and even death, particularly threatening vulnerable groups such as children and pregnant women. Frequent food safety incidents weaken consumer confidence and impact the food industry chain. Therefore, ensuring food safety is essential to maintain individual life rights, promote sustainable economic operation, and maintain social stability.

Just as people judge the degree of food spoilage by the smell of food, biosensors judge the state of food by detecting specific odors produced during food spoilage. Yang et al. produced nanodisks embedded with an OR produced in E. coli and then immobilized it on the floating electrodes of a carbon nanotube transistor [115]. Cadaverine as a death-associated odor was a sign of food spoilage (Figure 11a). Trace-amine-associated receptor 13c was employed to detect the target ligand. Study result indicated that this biosensor was sensitive to cadaverine stimulation as low as 10 pM. Calabrese et al. designed a non-Faradaic impedimetric biosensor for monitoring the presence of VOCs (1-octen-3-ol, trans-2-hexen-1-ol, and hexanal), involved in food spoilage [66]. This biosensor exploited pig OBP and could be used for measurements in 0.1 µM liquid and air samples.

Salmonella is a common foodborne pathogen that causes a series of symptoms such as typhoid or paratyphoid fever symptoms, bacterial blood symptoms, local infection symptoms, etc. In reference [181], researchers detected Salmonella contamination in packaged beef by measuring 3-methyl-1-butanol and 1-hexanol. The response signals from four QCMs were processed by principal component analysis and utilized for discriminating between these two odorants. Wang et al. immobilized silk fibroin microspheres onto lotus silk to form a high-surface-area mace structure and then functionalized it with Drosophila OBP-derived peptide [182]. The detection limit of this biosensor reached 1 ppt and sensed a 10⁵ CFU concentration of Salmonella in ham. Similar detection effects can be found in references [133,183] (Figure 11b).

Besides bacterial contamination, antibiotic abuse results in residues in animal-derived foods, which, when accumulated in the human body via the food chain, cause ototoxicity, nephrotoxicity, and neuromuscular blockade. Cui et al. proposed an aptamer probe that contained an aptamer unit for recognizing kanamycin, a trigger unit for triggering catalytic hairpin assembly and a suppression unit for ensuring the stability of the aptamer probe [184]. The kanamycin concentration was reflected as a fluorescent intensity with a detection limit of 0.26 ng/mL.

3.4. Security

Explosive detection is essential for maintaining public safety. By accurately identifying and intercepting explosive devices or hazardous chemicals, it effectively prevents casualties, property damage, and social panic. In airports, stations, and large events, rapid and sensitive detection technologies can screen suspicious items in real time and block potential threats.

Explosive detection via odor biosensors started from whole animals [185]. Animals such as dogs, pigs, mice, and honeybees could perform specific actions based on some explosive odors after training [186], thereby alerting security personnel to investigate potential hazards [187]. To save time and economic costs in training, Gao et al. implanted a microelectrode array into mouse olfactory bulb [58]. Since the mouse OSN M72 was sensitive to trinitrotoluene (Figure 12a), they used green fluorescent protein to highlight the corresponding section in the olfactory bulb. Then, the electrode probe could precisely be inserted into the correct area. OBP, OR proteins, or major urinary proteins have been shown to be more effective than in vivo strategies [102,188,189] (Figure 12b). These proteins have thus been extracted and purified for the measurement of various trace explosives or explosive residues.

4. Future Prospects

Biosensors have been developed for over four decades since the concept was first proposed in the 1980s. Their detection performance has also significantly improved in terms of sensitivity, selectivity, response time, sensing range, and so on. The number of sensing elements in biosensors has increased to enhance their detection capability [39,88]. Suitable sensing equipment and signal processing methods have been proposed to improve the devices’ signal-to-noise ratio [190,191]. Controlling a thin liquid layer or utilizing OBP as a molecular transporter has achieved both liquid- and gas-phase odorant detection [55,116,192].

Despite the fact that so many efforts have been made, odor biosensors’ performance is still far from that of animal olfactory systems. The lifetime of odor biosensors is hours or days, and, while in vivo biosensors are a promising solution, they would fail due to protein-wrapped electrodes [59,60]. The responses from biosensors usually become smaller or even invisible under continuous odor stimulation, which is the main barrier to long-term repeatable odor quantification [193]. Although some calibration or compensation strategies have been proposed [194], the problem of response attenuation has not been fundamentally solved yet. Variation in environmental factors, e.g., temperature, humidity, and light intensity, also influences biosensors’ response. Therefore, commercially available biosensors that can be deployed in industrial sites are quite rare. Moreover, the preparation of biological sensing materials is complex and expensive. Due to these limitations, biosensors are still mainly studied in the laboratory, with few successful application cases.

As for the future development of odor biosensors, higher sensitivity and selectivity and shorter response times are major objectives pursued by scientists. With the deepening of studies on animal olfaction, researchers have explored the response repertoires of different ORs and OBP. A database with more complete response information is required, as it would help users determine the optimal sensing material based on the target odorant. Moreover, peptides derived from various proteins inherit the detection performance of the original proteins and are easy to integrate into various transducers, making them ideal materials for rapid detection kits. We believe that, with the advancement of these technologies, commercial biosensors will soon be launched, thereby promoting biosensor research as a whole.

5. Conclusions

Odor biosensors, as an interdisciplinary technology which combines biometric elements with physical and chemical transducers, have shown broad application prospects in the environmental monitoring, disease diagnosis, food safety, and security fields due to their high specificity, sensitivity, and real-time response capabilities. By synergistically designing biological components such as OR proteins, OBPs, enzymes, antibodies, OSNs, or aptamers with electrochemical, optical, piezoelectric, and other transducers, these biosensors can accurately capture trace target odors and convert them into quantifiable signals, providing efficient solutions for dynamic detection in complex scenarios. In the future, with the deep integration of nanomaterials, flexible electronics, and artificial intelligence technologies, odor biosensors will develop towards miniature, intelligence, and multi-omics applications, further promoting their innovative use in precision medicine, intelligent environmental governance, and real-time risk warning, becoming the pioneers of “artificial olfaction” in order to safeguard human health and safety.