Eun Hae Oh

Polypyrrole nanotubes conjugated with human olfactory receptors: high-performance transducers for FET-type bioelectronic noses.

Get a whiff of this: Human olfactory receptor (hOR)-conjugated polypyrrole (PPy) nanotubes were integrated into the field-effect transistor (FET) sensor platform for the fabrication of high-performance bioelectronic noses (see picture, S = source, D = drain). The device can translate and amplify hOR-odorant interaction into a detectable signal, and it showed highly sensitive and specific responses toward a target odorant.

Recent advances in electronic and bioelectronic noses and their biomedical applications

Significant effort has been made in the development of an artificial nose system for various applications. Advances in sensor technology have facilitated the development of high-performance electronic and bioelectronic noses. Numerous articles describe the advantages of artificial nose systems for biomedical applications. Recent advances in the development of electronic and bioelectronic noses and their biomedical applications are reviewed in this article.

Nanovesicle-Based Bioelectronic Nose for the Diagnosis of Lung Cancer from Human Blood

A human nose-mimetic diagnosis system that can distinguish the odor of a lung cancer biomarker, heptanal, from human blood is presented. Selective recognition of the biomarker is mimicked in the human olfactory system. A specific olfactory receptor recognizing the chemical biomarker is first selected through screening a library of human olfactory receptors (hORs). The selected hOR is expressed on the membrane of human embryonic kidney (HEK)-293 cells. Nanovesicles containing the hOR on the membrane are produced from these cells, and are then used for the functionalization of single-walled carbon nanotubes. This strategy allows the development of a sensitive and selective nanovesicle-based bioelectronic nose (NvBN). The NvBN is able to selectively detect heptanal at a concentration as low as 1 × 10(-14) m, a sufficient level to distinguish the blood of a lung cancer patient from the blood of a healthy person. In actual experiments, NvBN could detect an extremely small increase in the amount of heptanal from human blood plasma without any pretreatment processes. This result offers a rapid and easy method to analyze chemical biomarkers from human blood in real-time and to diagnose lung cancer.

Cell-based high-throughput odorant screening system through visualization on a microwell array.

The development of a cell-based high-throughput screening system has attracted much attention from researchers who study drug screening mechanisms and characterization of G-protein coupled receptors (GPCRs). Although olfactory receptors (ORs) constitute the largest group of GPCRs that play a critical role recognizing and discriminating odorants, only a few ORs have been characterized, and most remain orphan. The conventional cell-based assay system for characterizing GPCRs, including ORs, is very laborious, time consuming, and requires an expensive assay system. In this study, we developed a simple, low-cost miniaturized odorant screening method by combining Micro-Electro-Mechanical system (MEMs) technique and visualization technique for detecting an odorant response. We fabricated PEG microwell from a photocrosslinkable polyethylene glycol diacrylate (PEGDA) solution and applied it to cell culture and a reverse transfection platform for cell-based high-throughput screening. For the first time, the olfactory receptors were expressed on the microwell platform using reverse transfection technique. The various olfactory receptors can be expressed simultaneously using this technique and the microwell spotted with olfactory receptor genes can be used as a high-throughput screening platform. The odorant response was detected via fluorescence analysis on the microwell using a cAMP response element (CRE) reporter assay. We tested this platform using four de-orphaned ORs. This new cell-based screening method not only reduced numerous time-consuming steps but also allowed for simple, efficient, and quantitative screening and patterning of large numbers of GPCRs including ORs, which can help to visualize the OR response to odorants on a microwell.

Specificity of odorant-binding proteins: a factor influencing the sensitivity of olfactory receptor-based biosensors

Odorant-binding proteins (OBPs) primarily function in the transport of hydrophobic odorants. In this study, OBPs originating from rat and pig were cloned into a mammalian expression vector, pcDNA3, and expressed in HEK-293 cells, and their specificity for odorants and olfactory receptors was examined. Results suggest that OBPs have a high affinity for the olfactory receptors when both the OBP and receptor originate from the same species. The rat OBPs were bound not only to the rat olfactory receptor I7 but also to the odorant specific to I7. The solubility of the odorant was increased by both OBP2 and OBP3, which originate from rat, but with different efficiencies. These results demonstrate that OBPs specifically interact with odorants as well as olfactory receptors, and these interactions can influence the sensitivity of olfactory receptor-based biosensors.

Cell-based microfluidic platform for mimicking human olfactory system

Various attempts have been made to mimic the human olfactory system using human olfactory receptors (hORs). In particular, OR-expressed cell-based odorant detection systems mimic the smell sensing mechanism of humans, as they exploit endogenous cellular signaling pathways. However, the majority of such cell-based studies have been performed in the liquid phase to maintain cell viability, and liquid odorants were used as detection targets. Here, we present a microfluidic device for the detection of gaseous odorants which more closely mimics the human olfactory system. Cells expressing hOR were cultured on a porous membrane. The membrane was then flipped over and placed between two compartments. The upper compartment is the gaseous part where gaseous odorants are supplied, while the lower compartment is the aqueous part where viable cells are maintained in the liquid medium. Using this simple microfluidic device, we were able to detect gaseous odorant molecules by a fluorescence signal. The fluorescence signal was generated by calcium influx resulting from the interaction between odorant molecules and the hOR. The system allowed detection of gaseous odorant molecules in real-time, and the findings showed that the fluorescence responses increased dose-dependently in the range of 0-2 ppm odorant. In addition, the system can discriminate among gaseous odorant molecules. This microfluidic system closely mimics the human olfactory system in the sense that the submerged cells detect gaseous odorants.

Coupling of olfactory receptor and ion channel for rapid and sensitive visualization of odorant response

In the human smell sensing system, there are about 390 kinds of olfactory receptors (ORs) which bind to various odorants with different affinities and specificities. Characterization and odorant binding pattern analysis of the ORs are essential for understanding of human olfaction and to mimic the olfactory system in various applications. Although various cell-based odorant screening systems have been developed for this purpose, many human ORs (hORs) still remain orphan because of the time-consuming and labor-intensive experimental procedures of the available screening methods. In this study, we constructed an ion channel-coupled hOR for simple odorant detection by rapidly visualizing the odorant response to overcome the limitations of conventional screening systems. The hORs were coupled to the Kir6.2 potassium channel and the fusion proteins were expressed in HEK293 cells. In this system, when an odorant binds to the hORs coupled to the ion channel, a conformational change in the OR occurs, which consequently opens the ion channel to result in ion influx into the cell. This ion influx was then visualized using a membrane potential dye. Cells expressing ion channel-coupled hORs showed high sensitivity and selectivity to their specific odorants, and the odorant-hOR binding pattern was visualized to identify the response of individual hORs to various odorants, as well as the response of various hORs to various odorants. These results indicate that the ion channel-coupled hOR system can be effectively used not only for simple and fast high-throughput odorant screening, but also to visualize the odorant-hOR response pattern.

Ion-Channel-Coupled Receptor-Based Platform for a Real-Time Measurement of G-Protein-Coupled Receptor Activities

A simple but efficient measurement platform based on ion-channel-coupled receptors and nanovesicles was developed for monitoring the real-time activity of G-protein-coupled receptors (GPCRs). In this work, an olfactory receptor (OR), the most common class A GPCR, was covalently fused with a Kir6.2 channel so that the GPCR action directly induced the opening of the ion channels and changes in the electrical membrane potential without complex cellular signaling processes. This strategy reduced the measurement errors caused by instability of various cellular components. In addition, rather than using whole cells, a cell-surface-derived nanovesicle was used to preserve the membrane-integrated structure of GPCRs and to exclude case-dependent cellular conditions. Another merit of using the nanovesicle is that nanovesicles can be easily combined with nanomaterial-based field-effect transistors (FETs) to build a sensitive and stable measurement platform to monitor GPCR activities with high sensitivity in real-time. Using a platform based on carbon nanotube FETs and nanovesicles carrying Kir6.2-channel-coupled ORs, we monitored the real-time response of ORs to their ligand molecules. Significantly, since this platform does not rely on rather unstable cell signaling pathways, our platform could be utilized for a rather long time period without losing its functionality. This system can be utilized extensively for simple and sensitive analysis of the activities of various GPCRs and should enable various academic and practical applications.

Applications and Perspectives of Bioelectronic Nose

Detection and discrimination of odorants has great potential for applications in various fields, such as the food industry, fragrance and flavor industry, environmental monitoring, and biomedical diagnosis. For several decades, many efforts have been made to control the process of food production and fragrance and flavor of brands, and to monitor environmental pollutions through the use of comparable technology. There have been several classical methods for these purposes. Conventional methods, such as GC/MS or human sensory panels (olfactometry), have been conventionally used, but they are expensive, labor-intensive, time-consuming and affected by large variations according to the conditions of analysis. These drawbacks increased the requirement for new technique, substituting classical methods, and the electronic nose has been developed over the past couple of decades. However, the electronic nose has also many limitations to be overcome. Recently, the bioelectronic nose, using biological components, has been developed. The bioelectronic nose has a bright prospect as a powerful and effective biosensing system, capable of detecting and discriminating a huge variety of odorant molecules. The most meaningful characteristics of the bioelectronic nose are that it mimics the human olfactory system. The bioelectronic nose is expected to replace the sensory evaluation method. It can be used for standardization of smell, development of code for each smell, and visualization of smell. Consequently, the development of the bioelectronic nose is expected to open up many new possibilities to improve the quality of our life.