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Electrochemical Signal Amplification in Olfactory and Taste Evaluation
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Biosensors are powerful analytical tools used to identify and detect target molecules. Electrochemical biosensors, which combine biosensing with electrochemical analysis techniques, are efficient analytical instruments that translate concentration signals into electrical signals, enabling the quantitative and qualitative analysis of target molecules. Electrochemical biosensors have been widely used in various fields of detection and analysis due to their high sensitivity, superior selectivity, quick reaction time, and inexpensive cost. However, the signal changes caused by interactions between a biological probe and a target molecule are very weak and difficult to capture directly by using detection instruments. Therefore, various signal amplification strategies have been proposed and developed to increase the accuracy and sensitivity of detection systems. 

electrochemical biosensors olfactory and taste evaluation signal amplification strategies nanomaterials enzymes nucleic acid amplification techniques
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Table of Contents

    1. Electrochemical Biosensors

    In the 1960s, Leland C. Clark Jr, an American scholar in electroanalytical chemistry, suggested that that the determination of biochemicals could be found using a method as convenient as pH electrodes, which led to the introduction of enzyme electrodes, the first biosensors [1][2][3]. For half a century, biosensing has developed into a classic converging technology with the incorporated principles and technologies of multiple disciplines such as life sciences, chemistry, physics, information, and materials [4]. In the 1970s to 1980s, various biomolecules and biomaterials were used as the molecular recognition elements for biosensors, enabling the rapid detection of a variety of biochemical and immunological substances [4]. In addition, various physical and chemical transduction principles were adopted, driving the formation of the biosensing field. In the second wave of development, second-generation enzyme electrodes were commercially successful [4], surface plasmon resonance (SPR) biosensors were widely used for biomolecular interaction studies [5], while DNA microarrays enabled high-throughput analysis of gene expression [4]. Since the 21st century, the introduction of nanotechnology has endowed biosensing with many new properties such as high sensitivity, a multiparameter nature, and microenvironmental applications [6]. Biosensors are powerful pieces of analytical equipment used to identify and detect target molecules, and are usually composed of a biosensing material and a physicochemical sensor [7]. Biosensors are generally used as detectors, and utilize a bioactive substance as a biofunctional sensitive element fixed to a signal transducer, which transmits a signal that is then converted to corresponding optical, thermal, and electrical signals with good sensitivity, selectivity, and specificity when a specific target is added. However, a bottleneck in the application of the biological receptor elements is the maintenance of their vitality, stability and shelf-life upon bonding with the electronic elements [8].
    Among the known types of biosensors, electrochemical biosensors are efficient analytical tools that combine biosensing and electrochemical analysis techniques [9] and are generally built in three-electrode electrochemical cells that consist of a working electrode, a counter electrode, and a standard electrode with a stable and fixed potential [10]. Analytical methods for electrochemical biosensors are usually based on the electron transfer process between an electrode surface and an electroactive material in an electrolyte [7]. Electrochemical biosensors use a fixed electrode as the base electrode and fixed bioactive molecules on their surfaces, capturing target molecules onto the electrode surface through specific recognition between biomolecules, where the base electrode converts the concentration signal into measurable electrical signals such as current, potential, and resistance. This enables the quantitative and qualitative analysis of a target. The basic principle of electrochemical biosensors is shown in Figure 1. Four signal conversion types exist for electrochemical biosensors: current, potential, impedance, and ion charge (field effect). Among the current-based electrochemical biosensors, commonly used detection methods include cyclic voltammetry (CV) [11], square wave voltammetry (SWV) [12], differential pulse voltammetry (DPV) [11], and electrochemical impedance spectroscopy (EIS) [12]. Electrochemical biosensors have been widely studied in simple or complex detection environments and in a variety of fields due to their high selectivity for the molecules they can identify, as well as their high sensitivity [13], fast response time, miniaturized and portable properties [14], compatibility with impurity matrices [15][16], simplicity of operation, and low cost [17][18].
    Figure 1. Basic principles of electrochemical biosensors.
    Although electrochemical biosensors are already highly sensitive, their sensitivity must be further improved for the detection of certain molecules at low concentrations or molecules that are difficult to isolate from biological samples [19][20][21][22]. In recent years, detection methods for specific interactions between biological recognition elements, such as antibodies, nucleotides, enzymes, and target analytes have been proposed and developed to improve the sensitivity and selectivity of detection systems [23]. In addition, signal amplification technology is often used as a critical technology in biosensor manufacturing because it plays a crucial role in improving the sensitivity, selectivity and stability of biosensors. Several signal amplification strategies, such as the use of nanomaterials with unique physicochemical properties, as well as the use of enzymatic labeling and nucleic acid amplification techniques, have become widespread. This entry will present several aspects of signal amplification strategies commonly used in electrochemical biosensors (Figure 2), as well as present recent results regarding their use in olfactory and taste determination.
    Figure 2. Signal amplification strategies commonly used in electrochemical biosensors.

    2. Advances in Electrochemical Signal Amplification Strategies for Olfactory and Taste Measurements

    The detection of gases, including malodorous molecules and volatile organic compounds (VOCs), has attracted great interest in recent years and there has been a growing demand for it in various fields. Volatile organic compounds (VOCs) are a large class of low molecular weight (<300 Da) carbon-containing compounds. These small volatile molecules have a wide range of sources, both natural (plants, animals, bacteria, etc.) and anthropogenic (fossil fuels, automobile exhaust, etc.) [24]. Studies have shown that most VOCs have adverse effects on human health, causing symptoms such as headaches, and nose, eye and throat irritation [25]. They are also considered chemical messengers, and studies have identified different gases associated with different diseases. In addition, VOC and odor analysis can be used for quality assessment in the food, beverage, and flavor industries. Therefore, it is crucial to monitor the nature and concentration of these compounds in indoor or outdoor environments [24].

    2.1. Classical Analytical Techniques for Olfactory and Taste Detection

    Olfactory and taste sensation are widespread in nature, and they play a major role in the survival and reproduction of natural organisms [26][27]. Olfactory and taste receptors mainly consist of cellular, tissue, or biological sensing receptors for various signals around the body, especially for food and its nutrients. Studies have shown that olfactory perception and taste are dependent on the sensing effect of G-protein-coupled receptors (GPCRs), making them the most important targets for drug screening. GPCRs are a superfamily of thousands of members that plays an extremely important role as nutrient sensor receptors in the metabolism of substances, capacity metabolism, and signal communication in the body or cells [28]. Methods commonly used for olfactory and taste detection include gas chromatography (GC), gas chromatography-mass spectrometry (GC-MS), electronic nose (EN), electronic tongue (ET) [29], near-infrared spectrum (NIR), and other biosensors based on olfactory receptors (OR) or taste receptors (TR) [30][31]. In addition, natural elements such as odor-binding protein (OBP) or its analogs, such as peptides, are often used in the construction of olfactory electrochemical biosensors [8][24][32][33].
    GC is a separation and analysis method using gas as a mobile phase, which has the advantages of high separation efficiency, fast analysis, high sensitivity and good selectivity, etc. [34]. It has been widely used in various fields and plays an important role in various aspects of modern society. GC consists of five systems: the gas circuit system, the sample injection system, the separation system, the temperature control system and the detection and recording system, of which the separation system and the detection and recording system are the core. With an inert gas as the mobile phase, GC takes advantage of the fact that the partition coefficient of components in a sample varies with the gas and stationary phases [34]. As the sample is carried into the column by the carrier gas, the components undergo repeated alternating distribution between the two phases. The components in the stationary phase have different absorption capacities and therefore the analytes pass through the column at different rates. After a certain column length, the components of the sample are separated from each other and enter the detector. The ion current signal generated by each component is amplified to produce a peak for each component. In this way, the purpose of separation and detection is achieved. Gas chromatography-mass spectrometry (GC-MS), a highly sensitive and accurate analytical technique, allows the separation, identification, and quantification of different VOCs in a mixture [34]. NIR is an electromagnetic spectrum between visible light (VIS) and mid-infrared (MIR). NIR is a method that uses chemical bonds containing hydrogen groups to stretch and relax frequencies, resulting in vibrational and combined frequencies. Fourier transform infrared spectroscopy (FTIR) requires a spectrometer and scanning means to analyze gases. FTIR can measure and analyze the concentration of toxic gases in a wide range of infrared regions [34].
    Although GC and GC-MS have good odor detection capabilities, they are not olfactory sensors. In addition, they are bulky and expensive and require highly time-consuming laboratory operations [34]. FTIR is highly sensitive and enables simultaneous analytical measurements of multiple gases, but its gas measurement and analysis can only be performed in the laboratory, thus making it impossible to achieve online real-time gas detection using FTIR [34]. Such a background has prompted many researchers to work on developing alternative techniques to overcome the various drawbacks mentioned above. Therefore, there is a need for an affordable, reliable, portable and sensitive device that can rapidly analyze gases, including VOCs [24].

    2.2. Olfactory and Taste Detection Based on Biosensor Technology

    Electronic noses and electronic tongues can detect odors and taste by using chemically sensitive materials and are based on chemical interactions [35][36][37]. They are fast, simple, and portable detection tools. However, they are unable to distinguish between chemicals with similar structures [37]. Linda B. Buck and Richard Axel have conducted numerous research efforts on biological olfaction, which has shown that in order to distinguish between various odors, biological noses exploit the cross-reactivity of olfactory receptors (OR), prompting each receptor to interact with different odor molecules [38].Thus, as with barcodes, odors are encoded by combinations of olfactory receptors, prompting the nose to have a wide detection range [24]. In addition, Linda B. Buck and Richard Axel demonstrated that ORs belong to the large family of G-protein-coupled receptors (GPCRs) [24]. In bioelectronic noses (B-EN) and bioelectronic tongues (B-ET), more selective odor detection can be achieved by using specific receptors, and transistor-based nanomaterials can be used to amplify sensory signals, such as carbon nanotubes [39], conductive polymers [40], and GR [41]. Bioelectronic noses-based nanomaterials are small, highly portable, and can be used for field odor analysis by combining them with portable current measurement devices [42]. In recent years, bioelectronic devices that use human sensory receptors as molecular recognition elements have been developed and have been commonly used to characterize food quality and safety. In addition, multi-channel bioelectronic noses have been developed that consist of arrays of olfactory receptors capable of individually analyzing various odor information [42][43]. However, most current EN systems use chemical layers as sensing elements and, therefore, have the disadvantage of limited diversity of sensor coatings and poor selectivity [24]. In addition, EN, B-EN, ET, and B-ET instruments not only have the disadvantage of a lack of sensor stability, but also the difficulty of having identical sensing characteristics of the instrument in different production batches. Recent research trends suggest that natural elements, such as ORs, OBP and peptides, can also be used as sensitive materials in biosensors to improve odor sensing performance [44][45].
    Since the discovery of the vertebrate olfactory receptor (ORs) gene family by Buck and Axel, much progress has been made in the study of the molecular mechanisms of olfaction and signal transduction pathways [31][38]. The process of biological olfactory is the selective recognition of odors by ORs, triggering the intracellular signal transduction pathways that lead to the depolarization of the OSN, and ultimately the transmission of information to the brain for processing via neuronal axonal connections. Olfactory biosensors use similar signal transduction mechanisms to recognize different odors and convert odor chemical signals into readable signals, such as electrical, and optical signals [31]. Technologies such as microelectrodes, light addressable potential sensors (LAPS), field effect transistors (FETs), and electrochemical impedance spectroscopy (EIS) have been commonly used in OR-based electrochemical biosensors for olfactory signal conversion. FETs have inherent signal amplification, making them particularly suitable for the detection of weak signals in OR-based biosensors [31].
    In biosensors, the characteristics and properties of the sensing material should be maintained by adopting an appropriate immobilization strategy, depending on the biometric parts. Methods that couple biological elements to appropriate transduction systems can often be used to obtain measurable and detectable signals [8]. However, combining ORs with signal transducers is a major challenge as ORIS membranes are bound to volatile organic compounds (VOCs), making it difficult to obtain configurations where ORs can bind to VOCs under membrane-free conditions [8]. To facilitate the incorporation of ORs into sensors, six materials have been typically utilized for binding, including cells and tissues, nanovesicles, nanodiscs, artificial lipid bilayers, odorant binding proteins, and biomimetic materials [8]. When embedding ORs using nanovesicles, the cell membrane containing an OR can be constructed as a nanoscale phospholipid bilayer structure to maintain the natural environment of the OR [46][47]. In addition, when the OR is integrated into a sensor using nanodiscs, these nanodiscs can provide a stable environment for ORs. However, this will separate the OR from the downstream proteins that promote the olfactory process [47]. Artificial lipid bilayers can simulate cell membranes, maintaining intact membrane proteins, such as ORs, and keeping their function intact. Biomimetic materials allow for the immobilization of synthetic peptides based on ORs and OBP rather than entire proteins, as they do not require tertiary structures or lipid membranes, thus improving stability and repeatability. Artificial receptors, such as molecularly imprinted polymers (MIP), can also be used as sensing elements due to their high stability [8].
    Compared to traditional odor analysis techniques, electronic noses, electronic tongues, and olfactory and gustatory electrochemical biosensors are fast, convenient, and economical, and are widely used in food, medicine, agriculture, and environmental monitoring [34]. In addition, gas sensing plays an important role in security applications (detection of drugs, explosives, etc.), environmental monitoring, and other applications under development, such as augmented or virtual reality [48]. Nevertheless, research on biosensors for olfaction and taste determination is still in the early experimental stages and further research is still needed as commercial OR biosensors are not yet available in the market due to the fragility of biosensing elements and the lack of portable signal transduction systems.

    2.3. Taste Electrochemical Sensors Based on a Cellular Signal Cascade Amplification System

    Currently, most studies that are focused on taste receptor sensors are based on changes in ion channels such as Ga2+ inward flow resulting from receptor–ligand interactions in living cells, which are dependent on a variety of complex factors such as cell type, physiological activity, environment, and intercellular interactions [34]. Although molecular interaction instruments based on SPR technology that can be used to detect non-standard receptor–ligand interactions are on the market, they are costly, technically complex, and struggle to achieve high throughput, making conventional taste detection difficult [34]. In current research, the methods for quantifying taste sensation have usually been based on three mechanisms, namely: labeling based on ion channels or cellular active components in living cells, non-standard-SPR methods, where the binding and dissociation properties of receptor–ligands are measured, and electronic tongues. In modern biotechnology, multiple molecular signal transduction components can be co-expressed in heterogeneous cell systems, thereby converting chemical signals into electrical signals [31].
    Lee et al. [49] designed a miniature planar electrode to record the general membrane potential changes of a heterogeneous olfactory system based on the co-expression of ORI7 and taste cyclic nucleotide gate (CNG) channels in HEK-293 cells. An olfactory biosensor based on the multipoint detection of the electrical activity of olfactory cells and tissues combined signal processing methods with olfactory decoding theory and showed excellent potential for the simultaneous detection of multiple odors in complex environments with high sensitivity and selectivity [31]. Xu et al. [50] constructed a novel hGPR120 fatty acid receptor sensor based on the self-assembly of the hGPR120 receptor onto the surface of bilayer-modified gold nanoparticles and bovine taste buds, which successfully detected the G protein signals generated by the interaction of this sensor with 14 different natural fatty acids. Pang et al. [34] self-assembled the T1R1 umami receptor protein expressed in vitro by rats onto nanogold, and constructed an electrochemical biosensor based on signal amplification using horseradish peroxidase for the quantitative determination of glutamate monosodium salt concentrations.
    An electrochemical taste sensor based on cell signal amplification has the advantages of high sensitivity, strong specificity, quantification, simple operation, low price, and good repeatability, and this technology can provide a good platform for the study of GPCRs and their interaction patterns with ligands and biological functions, which can then be used for taste determination in humans and animals [34].

    2.4. Olfactory Electrochemical Sensors Based on a Cellular Signal Cascade Amplification System

    Compared to other detection methods, OR electrochemical sensors based on the signal cascade amplification systems of the cell itself should be more sensitive by several orders of magnitude for detecting their respective ligands [51]. Studies have shown that ORCO expression will lead to an increase in ligand sensitivity and a decrease in the lower limit of detection [52]. The use of OR-expressing cells and tissues as biosensing elements is based on the generation of signal cascades of ions transferred from the outside to the inside of cells caused by the combination of OR odor [46]. In addition, OR electrochemical sensors can be coupled with enzymatic and nanomaterial electrochemical signal amplification methods. Given the natural diversity of the olfactory system and the compounds that can bind to gas molecules, a wider range of biosensors can be constructed for different application aspects [8]. Lu et al. [31] prepared a sandwich electrochemical olfactory sensor to detect sex differences in male and female rats, using the vomeronasal organ tissue of rats as a reference. The results showed that the vomeronasal organ sensors of male and female rats had different dynamic curves for their respective urine and were able to distinguish between their own urine and the urine from other rats. Lu et al. [53] simulated intracellular receptor signal processes based on an electrochemical signal amplification system of gold nanoparticles (AuNPs) and HRP. Using gold nanoparticles self-assembled twice and the subsequent adsorption of Bombyx olfactory receptor 1 (BmOR1), a sex pheromone binding protein, an electrochemical upper nanogold membrane receptor sensor was constructed. Kang et al. [54] constructed an H2O2 electrochemical biosensor based on the nanogold adsorption of immobilized horseradish peroxidase HRP with thionine-chitosan as a bridging agent. On this basis, a bilayer nanogold-modified Bacillus cereus immunosensor was prepared based on the nanogold adsorption of HRP for electrochemical signal amplification using Bacillus cereus monoclonal antibody as the biomolecular recognition element and chitosan as the bridging agent [55][56].
    In recent years, there have been tremendous advances in conductor technology, nanomaterials, carbon nanotubes, and GR, which have had some impact on the quality of signals obtained from electrochemical biosensors and their process improvements. Thus, various biometric materials based on olfactory sensing elements are expected to eventually be used to construct more sensitive and ultra-selective nanobiosensors by integrating them with various nanomaterials [8]. Biosensors based on OR have great potential for development and have promising applications in numerous fields due to their high sensitivity and specificity. For example, they can be applied to drug discovery by detecting interactions between ORs and drugs, as well as detecting specific interactions between ORs and odor substances, providing a useful platform for basic olfactory research. However, this research is still at an early experimental stage, and commercial OR biosensors are not yet available on the market due to the fragility of the biosensing components and the lack of small, portable signal transduction systems. As researchers gain a better understanding of odor binding sites, synthetic proteins, and peptides with higher stability and reliability, these will likely replace tissues and cells for odor detection. In addition, microfabrication technology improvement will also accelerate the miniaturization of OR-based biosensors, and synthetic biology will likely facilitate their further development. Thus, with the development and combination of multiple disciplines, commercial OR biosensors are bound to emerge and show promising applications in many fields of application [31].

    3. Commonly Used Signal Amplification Strategies for Electrochemical Biosensors

    3.1. Signal Amplification Strategies Based on Nanomaterials

    Nanomaterials are a type of material of an at least one-dimensional nanometer size (1–100 nm) in a three-dimensional space, and are characterized by their high electrical conductivity, good chemical stability, large specific surface area, and structural flexibility. Nanomaterials are endowed with unique surfaces, quantum size, and have been found to manifest macroscopic quantum tunneling effects [57], as well as having unique electronic and optical properties [58].
    Nanomaterials allow direct contact with a sensing environment, enabling rapid signal conduction and thus increasing system sensitivity and reducing detection limits [59]. Nanomaterials have been commonly used as carriers or capture carriers to immobilize a large number of markers (e.g., antibodies, nucleic acids, and enzymes) based on their unique properties, such as their nanostructures or superparamagnetic activities [60]. In addition, nanomaterials have been used as novel luminescent reagents to enhance signals by modulating the luminescence of nanomaterials, such as by adjusting their size or ligands [61], thereby enabling signal amplification. Among the common nanomaterials, metal nanomaterials (e.g., gold and silver nanomaterials), carbon nanomaterials, quantum dots, and metal-organic frameworks can be directly used as electroactive substances to achieve signal amplification in sensors [62]Table 1 lists some of the applications of nanomaterial-based signal amplification electrochemical biosensors for practical detection.


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      Wang, X.;  Lu, D.;  Liu, Y.;  Wang, W.;  Ren, R.;  Li, M.;  Liu, D.;  Liu, Y.;  Liu, Y.;  Pang, G. Electrochemical Signal Amplification in Olfactory and Taste Evaluation. Encyclopedia. Available online: (accessed on 02 June 2023).
      Wang X,  Lu D,  Liu Y,  Wang W,  Ren R,  Li M, et al. Electrochemical Signal Amplification in Olfactory and Taste Evaluation. Encyclopedia. Available at: Accessed June 02, 2023.
      Wang, Xinqian, Dingqiang Lu, Yuan Liu, Wenli Wang, Ruijuan Ren, Ming Li, Danyang Liu, Yujiao Liu, Yixuan Liu, Guangchang Pang. "Electrochemical Signal Amplification in Olfactory and Taste Evaluation" Encyclopedia, (accessed June 02, 2023).
      Wang, X.,  Lu, D.,  Liu, Y.,  Wang, W.,  Ren, R.,  Li, M.,  Liu, D.,  Liu, Y.,  Liu, Y., & Pang, G. (2022, August 18). Electrochemical Signal Amplification in Olfactory and Taste Evaluation. In Encyclopedia.
      Wang, Xinqian, et al. "Electrochemical Signal Amplification in Olfactory and Taste Evaluation." Encyclopedia. Web. 18 August, 2022.