Jong Hyun Lim

A peptide receptor-based bioelectronic nose for the real-time determination of seafood quality.

We herein report a peptide receptor-based bioelectronic nose (PRBN) that can determine the quality of seafood in real-time through measuring the amount of trimethylamine (TMA) generated from spoiled seafood. The PRBN was developed using single walled-carbon nanotube field-effect transistors (SWNT-FETs) functionalized with olfactory receptor-derived peptides (ORPs) which can recognize TMA and it allowed us to sensitively and selectively detect TMA in real-time at concentrations as low as 10fM. Utilizing these properties, we were able to not only determine the quality of three kinds of seafood (oyster, shrimp, and lobster), but were also able to distinguish spoiled seafood from other types of spoiled foods without any pretreatment processes. Especially, the use of small synthetic peptide rather than the whole protein allowed PRBNs to be simply manufactured through a single-step process and to be reused with high reproducibility due to no requirement of lipid bilayers. Furthermore, the PRBN was produced on a portable scale making it effectively useful for the food industry where the on-site measurement of seafood quality is required.

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.

Bioelectronic nose combined with a microfluidic system for the detection of gaseous trimethylamine

A bioelectronic nose based on a novel microfluidic system (μBN) was fabricated to detect gaseous trimethylamine (TMA) in real-time. Single-walled carbon nanotube-field effect transistors (SWNT-FETs) were functionalized with olfactory receptor-derived peptides (ORPs) that can recognize the TMA molecules. The ORP-coated SWNT-FETs were assembled with a microfluidic channel and were sealed with top and bottom frames. This simple process was used to complete the μBNs, and a well-defined condition was achieved to detect the gaseous molecules. The μBNs allowed us to detect gaseous TMA molecules down to 10 parts per trillion (ppt) in real-time and showed high selectivity when distinguishing gaseous TMA from other gaseous odorants. The sensor was used to determine the quality of seafood (oysters), and spoiled seafood and other types of spoiled foods were also successfully discriminated without any pretreatment processes. These results indicate that portable-scale platforms can be manufactured by using μBNs and can be applicable for real-time on-site gas analysis.

Real-time monitoring of geosmin and 2-methylisoborneol, representative odor compounds in water pollution using bioelectronic nose with human-like performance.

A bioelectronic nose for the real-time assessment of water quality was constructed with human olfactory receptor (hOR) and single-walled carbon nanotube field-effect transistor (swCNT-FET). Geosmin (GSM) and 2-methylisoborneol (MIB), mainly produced by bacteria, are representative odor compounds and also indicators of contamination in the water supply system. For the screening of hORs which respond to these compounds, we performed CRE-luciferase assays of the two odorants in heterologous cell system. Human OR51S1 for GSM and OR3A4 for MIB were selected, and nanovesicles expressing the hORs on surface were produced from HEK-293 cell. Carbon nanotube field-effect transistor was functionalized with the nanovesicles. The bioelectronic nose was able to selectively detect GSM and MIB at concentrations as low as a 10 ng L(-1). Furthermore, detection of these compounds from the real samples such as tap water, bottled water and river water was available without any pretreatment processes.

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.

Olfactory Receptor Screening Assay Using Nanovesicle-Immobilized Carbon Nanotube Transistor

Olfactory receptor (OR) genes are considered to be the largest superfamily of the mammalian genome, and in the case of humans, approximately 390 kinds of functional ORs play a role in perceiving odors. In spite of their significance in olfaction, the function of all ORs has not yet been fully revealed. In order to efficiently identify specific ligands of orphan ORs, methods that can generate olfactory signals in a reliable manner and that can convert the cellular signals into measurable responses are required. Here, we describe an OR screening assay method using olfactory sensors that are based on cell-derived nanovesicles combined with single-walled carbon nanotube field-effect transistors (SWNT-FETs). The nanovesicles contain ORs on their surface membrane and induce influx of calcium ions similar to olfactory signal transduction. This ion influx causes an electrical current change along the carbon nanotube, and then this change is measured by the SWNT-FET sensor. This technique facilitates the simple and rapid screening of OR functions.