Bioelectronic tongue using heterodimeric human taste receptor for the discrimination of sweeteners with human-like performance.

The sense of taste helps humans to obtain information and form a picture of the world by recognizing chemicals in their environments. Over the past decade, large advances have been made in understanding the mechanisms of taste detection and mimicking its capability using artificial sensor devices. However, the detection capability of previous artificial taste sensors has been far inferior to that of animal tongues, in terms of its sensitivity and selectivity. Herein, we developed a bioelectronic tongue using heterodimeric human sweet taste receptors for the detection and discrimination of sweeteners with human-like performance, where single-walled carbon nanotube field-effect transistors were functionalized with nanovesicles containing human sweet taste receptors and used to detect the binding of sweeteners to the taste receptors. The receptors are heterodimeric G-protein-coupled receptors (GPCRs) composed of human taste receptor type 1 member 2 (hTAS1R2) and human taste receptor type 1 member 3 (hTAS1R3), which have multiple binding sites and allow a human tongue-like broad selectivity for the detection of sweeteners. This nanovesicle-based bioelectronic tongue can be a powerful tool for the detection of sweeteners as an alternative to labor-intensive and time-consuming cell-based assays and the sensory evaluation panels used in the food and beverage industry. Furthermore, this study also allows the artificial sensor to exam the functional activity of dimeric GPCRs.

Functional characterization of naturally occurring melittin peptide isoforms in two honey bee species, Apis mellifera and Apis cerana.

Insect-derived antimicrobial peptides (AMPs) have diverse effects on antimicrobial properties and pharmacological activities such as anti-inflammation and anticancer properties. Naturally occurring genetic polymorphism have a direct and/or indirect influence on pharmacological effect of AMPs, therefore information on single nucleotide polymorphism (SNP) occurring in natural AMPs provides an important clue to therapeutic applications. Here we identified nucleotide polymorphisms in melittin gene of honey bee populations, which is one of the potent AMP in bee venoms. We found that the novel SNP of melittin gene exists in these two honey bee species, Apis mellifera and Apis cerana. Nine polymorphisms were identified within the coding region of the melittin gene, of which one polymorphism that resulted in serine (Ser) to asparagine (Asp) substitution that can potentially effect on biological activities of melittin peptide. Serine-substituted melittin (Mel-S) showed more cytotoxic effect than asparagine-substituted melittin (Mel-N) against E. coli. Also, Mel-N and Mel-S had different inhibitory effects on the production of inflammatory factors such as IL-6 and TNF-α in BV-2 cells. Moreover, Mel-S showed stronger cytotoxic activities than Mel-N peptide against two human ovarian cancer cell lines. Using carbon nanotube-based transistor, we here characterized that Mel-S interacted with small unilamellar liposomes more strongly than Mel-N. Taken together, our present study demonstrates that there exist different characteristics of the gene frequency and the biological activities of the melittin peptide in two honey bee species, Apis mellifera and A. cerana.

Graphene nanonet for biological sensing applications.

We report a simple but efficient method to fabricate versatile graphene nanonet (GNN)-devices. In this method, networks of V2O5 nanowires (NWs) were prepared in specific regions of single-layer graphene, and the graphene layer was selectively etched via a reactive ion etching method using the V2O5 NWs as a shadow mask. The process allowed us to prepare large scale patterns of GNN structures which were comprised of continuous networks of graphene nanoribbons (GNRs) with chemical functional groups on their edges. The GNN can be easily functionalized with biomolecules for fluorescent biochip applications. Furthermore, electrical channels based on GNN exhibited a rather high mobility and low noise compared with other network structures based on nanostructures such as carbon nanotubes, which was attributed to the continuous connection of nanoribbons in GNN structures. As a proof of concept, we built DNA sensors based on GNN channels and demonstrated the selective detection of DNA. Since our method allows us to prepare high-performance networks of GNRs over a large surface area, it should open up various practical biosensing applications.

Highly selective and sensitive detection of neurotransmitters using receptor-modified single-walled carbon nanotube sensors.

We present receptor-modified carbon nanotube sensors for the highly selective and sensitive detection of acetylcholine (ACh), one kind of neurotransmitter. Here, we successfully expressed the M1 muscarinic acetylcholine receptor (M1 mAChR), a family of G protein-coupled receptors (GPCRs), in E. coli and coated single-walled carbon nanotube (swCNT)-field effect transistors (FETs) with lipid membrane including the receptor, enabling highly selective and sensitive ACh detection. Using this sensor, we could detect ACh at 100 pM concentration. Moreover, we showed that this sensor could selectively detect ACh among other neurotransmitters. This is the first demonstration of the real-time detection of ACh using specific binding between ACh and M1 mAChR, and it may lead to breakthroughs for various applications such as disease diagnosis and drug screening.

"Chemical-pain sensor" based on nanovesicle-carbon nanotube hybrid structures.

We developed a "chemical-pain sensor" that could recognize chemical pain stimuli such as capsaicin and resiniferatoxin just like mammalian chemical pain sensory systems. Here, we first prepared nanovesicles containing rat pain sensory receptor, rat transient receptor potential vanilloid 1 (rTRPV1), which is activated by noxious heat and capsaicin. And the nanovesicles were immobilized on a single-walled carbon nanotube-based field effect transistor. The chemical-pain sensor could selectively detect chemical pain stimuli with a high sensitivity of a 1 pM detection limit. It also responded to different chemical pain stimuli in a manner similar as to that of mammalian chemical pain sensory systems. This sensor platform can be utilized for various practical applications such as food screening tools and artificial somesthetic sensors. Moreover, TRP families have been suggested as potential drug targets related to nerve and circulation disorders. Thus, the capability of monitoring TRP responses using our sensor platforms should provide a powerful means for the development of new drugs as well as the basic research about nerve and circulation systems.

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 platform mimicking human olfactory signal transduction.

We developed a nanovesicle-based bioelectronic nose (NBN) that could recognize a specific odorant and mimic the receptor-mediated signal transmission of human olfactory systems. To build an NBN, we combined a single-walled carbon nanotube-based field effect transistor with cell-derived nanovesicles containing human olfactory receptors and calcium ion signal pathways. Importantly, the NBN took advantages of cell signal pathways for sensing signal amplification, enabling ≈ 100 times better sensitivity than that of previous bioelectronic noses based on only olfactory receptor protein and carbon nanotube transistors. The NBN sensors exhibited a human-like selectivity with single-carbon-atomic resolution and a high sensitivity of 1 fM detection limit. Moreover, this sensor platform could mimic a receptor-meditated cellular signal transmission in live cells. This sensor platform can be utilized for the study of molecular recognition and biological processes occurring at cell membranes and also for various practical applications such as food screening and medical diagnostics.

A bioelectronic sensor based on canine olfactory nanovesicle-carbon nanotube hybrid structures for the fast assessment of food quality.

We developed an olfactory-nanovesicle-fused carbon-nanotube-transistor biosensor (OCB) that mimics the responses of a canine nose for the sensitive and selective detection of hexanal, an indicator of the oxidation of food. OCBs allowed us to detect hexanal down to 1 fM concentration in real-time. Significantly, we demonstrated the detection of hexanal with an excellent selectivity capable of discriminating hexanal from analogous compounds such as pentanal, heptanal, and octanal. Furthermore, we successfully detected hexanal in spoiled milk without any pretreatment processes. Considering these results, our sensor platform should offer a new method for the assessment of food quality and contribute to the development of portable sensing devices.

Nanotube-bridged wires with sub-10 nm gaps.

We report a simple but efficient method to synthesize carbon nanotube-bridged wires (NBWs) with gaps as small as 5 nm. In this method, we have combined a strategy for assembling carbon nanotubes (CNTs) inside anodized aluminum oxide pores and the on-wire lithography technique to fabricate CNT-bridged wires with gap sizes deliberately tailored over the 5-600 nm range. As a proof-of-concept demonstration of the utility of this architecture, we have prepared NBW-based chemical and biosensors which exhibit higher analyte sensitivity (lower limits of detection) than those based on planar CNT networks. This observation is attributed to a greater surface-to-volume ratio of CNTs in the NBWs than those in the planar CNT devices. Because of the ease of synthesis and high yield of NBWs, this technique may enable the further incorporation of CNT-based architectures into various nanoelectronic and sensor platforms.

Floating electrode transistor based on purified semiconducting carbon nanotubes for high source-drain voltage operation.

We report floating-electrode-based thin-film transistors (F-TFTs) based on a purified semiconducting single-walled carbon nanotube (swCNT) network for a high source-drain voltage operation. At a high source-drain voltage, a conventional swCNT-TFT exhibited poor transistor performance with a small on-off ratio, which was attributed to the reduced Schottky barrier modulation at a large bias. In the F-TFT device, an swCNT network channel was separated into a number of channels connected by floating electrodes. The F-TFTs exhibited a much higher on-off ratio than a conventional swCNT-TFT with a single channel. This work should provide an important guideline in designing swCNT-TFTs for high voltage applications such as displays.

Bioelectronic nose with high sensitivity and selectivity using chemically functionalized carbon nanotube combined with human olfactory receptor.

Single-walled carbon nanotubes (swCNTs) hold great promise for use as molecular wires because they exhibit high electrical conductivity and chemical stability. However, constructing swCNT-based transducer devices requires controlled strategies for assembling biomolecules on swCNTs. In this study, we proposed a chemically modified swCNT. The swCNT was functionalized with 1,5-diaminonaphthalene via π-stacking, for reliable attachment of the human olfactory receptor 2AG1 (hOR2AG1). The human olfactory receptor was then anchored. We investigated the use of this functionalized CNT in the fabrication of a highly sensitive and selective bioelectronic nose. For the bioelectronic nose, the swCNT-field effect transistor (FET) platform was composed of polyethylene glycol (PEG)-coated regions to prevent non-specific absorption and chemically modified swCNTs regions containing hOR2AG1, which can bind to the specific odorant. This approach allowed us to create well-defined micron-scale patterns of hOR2AG1 on the swCNTs. Our bioelectronic nose displayed ultrahigh sensitivity down to concentrations as low as 1fM due to the enhanced hOR2AG1-odorant interaction through the tight binding of hOR2AG1 on the chemically modified swCNTs. In addition, the approach described here may provide an alternative route for multiplexed detection of diverse odorants and to improve the sensitivity of sensor devices.

Polarization-controlled differentiation of human neural stem cells using synergistic cues from the patterns of carbon nanotube monolayer coating.

We report a method for selective growth and structural-polarization-controlled neuronal differentiation of human neural stem cells (hNSCs) into neurons using carbon nanotube network patterns. The CNT patterns provide synergistic cues for the differentiation of hNSCs in physiological solution and an optimal nanotopography at the same time with good biocompatibility. We demonstrated a polarization-controlled neuronal differentiation at the level of individual NSCs. This result should provide a stable and versatile platform for controlling the hNSC growth because CNT patterns are known to be stable in time unlike commonly used organic molecular patterns.

"Bioelectronic super-taster" device based on taste receptor-carbon nanotube hybrid structures.

We have developed a method to monitor the activities of human taste receptor protein in lipid membrane using carbon nanotube transistors, enabling a "bioelectronic super-taster (BST)", a taste sensor with human-tongue-like selectivity. In this work, human bitter taste receptor protein expressed in E. coli was immobilized on a single-walled carbon nanotube field effect transistor (swCNT-FET) with the lipid membrane. Then, the protein binding activity was monitored using the underlying swCNT-FET, leading to the operation as a BST device. The fabricated BST device could detect bitter tastants at 100 fM concentrations and distinguish between bitter and non-bitter tastants with similar chemical structures just like a human tongue. Furthermore, this strategy was utilized to differentiate the responses of taster or non-taster types of the bitter taste receptor proteins.