MXene/Hydrogel-based bioelectronic nose for the direct evaluation of food spoilage in both liquid and gas-phase environments.

Various bioelectronic noses have been recently developed for mimicking human olfactory systems. However, achieving direct monitoring of gas-phase molecules remains a challenge for the development of bioelectronic noses due to the instability of receptor and the limitations of its surrounding microenvironment. Here, we report a MXene/hydrogel-based bioelectronic nose for the sensitive detection of liquid and gaseous hexanal, a signature odorant from spoiled food. In this study, a conducting MXene/hydrogel structure was formed on a sensor via physical adsorption. Then, canine olfactory receptor 5269-embedded nanodiscs (cfOR5269NDs) which could selectively recognize hexanal molecules were embedded in the three-dimensional (3D) MXene/hydrogel structures using glutaraldehyde as a linker. Our MXene/hydrogel-based bioelectronic nose exhibited a high selectivity and sensitivity for monitoring hexanal in both liquid and gas phases. The bioelectronic noses could sensitively detect liquid and gaseous hexanal down to 10-18 M and 6.9 ppm, and they had wide detection ranges of 10-18 - 10-6 M and 6.9-32.9 ppm, respectively. Moreover, our bioelectronic nose allowed us to monitor hexanal levels in fish and milk. In this respect, our MXene/hydrogel-based bioelectronic nose could be a practical strategy for versatile applications such as food spoilage assessments in both liquid and gaseous systems.

Highly Sensitive Real-Time Monitoring of Adenosine Receptor Activities in Nonsmall Cell Lung Cancer Cells Using Carbon Nanotube Field-Effect Transistors.

Adenosine metabolism through adenosine receptors plays a critical role in lung cancer biology. Although recent studies showed the potential of targeting adenosine receptors as drug targets for lung cancer treatment, conventional methods for investigating receptor activities often suffer from various drawbacks, including low sensitivity and slow analysis speed. In this study, adenosine receptor activities in nonsmall cell lung cancer (NSCLC) cells were monitored in real time with high sensitivity through a carbon nanotube field-effect transistor (CNT-FET). In this method, we hybridized a CNT-FET with NSCLC cells expressing A2A and A2B adenosine receptors to construct a hybrid platform. This platform could detect adenosine, an endogenous ligand of adenosine receptors, down to 1 fM in real time and sensitively discriminate adenosine among other nucleosides. Furthermore, we could also utilize the platform to detect adenosine in complicated environments, such as human serum. Notably, our hybrid platform allowed us to monitor pharmacological effects between adenosine and other drugs, including dipyridamole and theophylline, even in human serum samples. These results indicate that the NSCLC cell-hybridized CNT-FET can be a practical tool for biomedical applications, such as the evaluation and screening of drug-candidate substances.

Plasmonic-Magnetic Active Nanorheology for Intracellular Viscosity.

We demonstrate active plasmonic systems where plasmonic signals are repeatedly modulated by changing the orientation of nanoprobes under an external magnetic field, which is a prerequisite for in situ active nanorheology in intracellular viscosity measurements. Au/Ni/Au nanorods act as "nanotransmitters", which transmit the mechanical motion of nanorods to an electromagnetic radiation signal as a periodic sine function. This fluctuating optical response is transduced to frequency peaks via Fourier transform surface plasmon resonance (FTSPR). As a driving frequency of the external magnetic field applied to the Au/Ni/Au nanorods increases and reaches above a critical threshold, there is a transition from the synchronous motion of nanorods to asynchronous responses, leading to the disappearance of the FTSPR peak, which allows us to measure the local viscosity of the complex fluids. Using this ensemble-based method with plasmonic functional nanomaterials, we measure the intracellular viscosity of cancer cells and normal cells in a reliable and reproducible manner.

Bioelectrical Nose Platform Using Odorant-Binding Protein as a Molecular Transporter Mimicking Human Mucosa for Direct Gas Sensing.

Recently, various bioelectronic nose devices based on human receptors were developed for mimicking a human olfactory system. However, such bioelectronic nose devices could operate in an aqueous solution, and it was often very difficult to detect insoluble gas odorants. Here, we report a portable bioelectronic nose platform utilizing a receptor protein-based bioelectronic nose device as a sensor and odorant-binding protein (OBP) as a transporter for insoluble gas molecules in a solution, mimicking the functionality of human mucosa. Our bioelectronic nose platform based on I7 receptor exhibited dose-dependent responses to octanal gas in real time. Furthermore, the bioelectronic platforms with OBP exhibited the sensor sensitivity improved by ∼100% compared with those without OBP. We also demonstrated the detection of odorant gas from real orange juice and found that the electrical responses of the devices with OBP were much larger than those without OBP. Since our bioelectronic nose platform allows us to directly detect gas-phase odorant molecules including a rather insoluble species, it could be a powerful tool for versatile applications and basic research based on a bioelectronic nose.

Ion-Selective Carbon Nanotube Field-Effect Transistors for Monitoring Drug Effects on Nicotinic Acetylcholine Receptor Activation in Live Cells.

We developed ion-selective field-effect transistor (FET) sensors with floating electrodes for the monitoring of the potassium ion release by the stimulation of nicotinic acetylcholine receptors (nAChRs) on PC12 cells. Here, ion-selective valinomycin-polyvinyl chloride (PVC) membranes were coated on the floating electrode-based carbon nanotube (CNT) FETs to build the sensors. The sensors could selectively measure potassium ions with a minimum detection limit of 1 nM. We utilized the sensor for the real-time monitoring of the potassium ion released from a live cell stimulated by nicotine. Notably, this method also allowed us to quantitatively monitor the cell responses by agonists and antagonists of nAChRs. These results suggest that our ion-selective CNT-FET sensor has potential uses in biological and medical researches such as the monitoring of ion-channel activity and the screening of drugs.

Nafion-Radical Hybrid Films on Carbon Nanotube Transistors for Monitoring Antipsychotic Drug Effects on Stimulated Dopamine Release.

We developed floating electrode-based carbon nanotube biosensors for the monitoring of antipsychotic drug effects on the dopamine release from PC12 cells under potassium stimulation. Here, carbon nanotube field-effect transistors with floating electrodes were functionalized with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•) radicals by Nafion films. This method allows us to build selective biosensors for dopamine detection with a detection limit down to 10 nM even in the presence of other neurotransmitters such as glutamate and acetylcholine, resulting from the selective interaction between ABTS• radicals and dopamine. The sensors were also utilized to monitor the real-time release of dopamine from PC12 cells upon the stimulation of high-concentrated potassium solutions. Significantly, the antipsychotic effects of pimozide on the dopamine release from potassium-stimulated PC12 cells could also be evaluated in a concentration-dependent manner by using the sensors. The quantitative and real-time evaluation capability of our strategy should provide a versatile tool for many biomedical studies and applications.

Dye-functionalized Sol-gel Matrix on Carbon Nanotubes for Refreshable and Flexible Gas Sensors.

We report a colorimetric dye-functionalized sol-gel matrix on carbon nanotubes for use as a refreshable and flexible gas sensor with humidity calibration. Here, we fabricated gas sensors by functionalizing dye molecules on the top of carbon nanotube networks via a sol-gel method. Using hybrid gas sensors with different dye molecules, we could selectively detect various hazardous gases, such as NH3, Cl2 and SO2 gases, via optical and electrical signals. The sensors exhibited rather large conductance changes of more than 50% following exposure to gas species with concentrations even under the permissible exposure limit. Significantly, we could refresh used gas sensors by simply exposing them to fresh N2 gas without any heat treatment. Additionally, our sensors can be bent to form versatile practical sensor devices, such as tube-shape sensors for ventilation tubes. This work shows a simple but powerful method for building refreshable and selective gas sensors for versatile industrial and academic applications.

Modified Floating Electrode-Based Sensors for the Quantitative Monitoring of Drug Effects on Cytokine Levels Related with Inflammatory Bowel Diseases.

Modified floating electrode-based sensors were developed to quantitatively monitor the levels of tumor necrosis factor α (TNF-α), a pro-inflammatory cytokine related with inflammatory bowel disease (IBD), and to evaluate the effect of drugs on the cytokine levels. Here, antibodies (anti-TNF-α) were immobilized on the floating electrodes of carbon nanotube devices, enabling selective and real-time detection of TNF-α among various cytokines linked to IBD. This sensor was able to measure the concentrations of TNF-α with a detection limit of 1 pg/L, allowing the quantitative estimation of TNF-α secretion from mouse macrophage Raw 264.7 cells stimulated by lipopolysaccharides (LPS). Notably, this method also allowed us to monitor the anti-inflammatory effect of a drug, lupeol, on the activation of the LPS-induced nuclear factor κB signaling in Raw 264.7 cells. These results indicate that our novel TNF sensor can be a versatile tool for biomedical research and clinical applications such as screening drug effects and monitoring inflammation levels.

Bioelectronic Nose Using Odorant Binding Protein-Derived Peptide and Carbon Nanotube Field-Effect Transistor for the Assessment of Salmonella Contamination in Food.

Salmonella infection is the one of the major causes of food borne illnesses including fever, abdominal pain, diarrhea, and nausea. Thus, early detection of Salmonella contamination is important for our healthy life. Conventional detection methods for the food contamination have limitations in sensitivity and rapidity; thus, the early detection has been difficult. Herein, we developed a bioelectronic nose using a carbon nanotube (CNT) field-effect transistor (FET) functionalized with Drosophila odorant binding protein (OBP)-derived peptide for easy and rapid detection of Salmonella contamination in ham. 3-Methyl-1-butanol is known as a specific volatile organic compound, generated from the ham contaminated with Salmonella. We designed and synthesized the peptide based on the sequence of the Drosophila OBP, LUSH, which specifically binds to alcohols. The C-terminus of the synthetic peptide was modified with three phenylalanine residues and directly immobilized onto CNT channels using the π-π interaction. The p-type properties of FET were clearly maintained after the functionalization using the peptide. The biosensor detected 1 fM of 3-methyl-1-butanol with high selectivity and successfully assessed Salmonella contamination in ham. These results indicate that the bioelectronic nose can be used for the rapid detection of Salmonella contamination in food.

"Bio-switch Chip" Based on Nanostructured Conducting Polymer and Entrapped Enzyme.

We report a switchable biochip strategy where enzymes were entrapped in conducting polymer layers and the enzymatic reaction of the entrapped enzymes was controlled in real-time via electrical stimuli on the polymer layers. This device is named here as a "bio-switch chip" (BSC). We fabricated BSC structures using polypyrrole (Ppy) with entrapped glucose oxidase (GOx) and demonstrated the switching of glucose oxidation reaction in real-time. We found that the introduction of a negative bias voltage on the BSC structure resulted in the enhanced glucose oxidation reaction by more than 20 times than that without a bias voltage. Moreover, because the BSC structures could be fabricated on specific regions, we could control the enzymatic reaction on specific regions. In view of the fact that enzymes enable very useful and versatile biochemical reactions, the ability to control the enzymatic reactions via conventional electrical signals could open up various applications in the area of biochips and other biochemical industries.

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.

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.

Reusable floating-electrode sensor for the quantitative electrophysiological monitoring of a nonadherent cell.

We report a reusable floating-electrode sensor based on aligned semiconducting single-walled carbon nanotubes for the quantitative monitoring of the electrophysiological responses from a nonadherent cell. This method allowed us to monitor and distinguish the real-time responses from normal and small-cell lung cancer (SCLC) cells to the addition of nicotine. The difference was attributed to the overexpressed nicotinic acetylcholine receptors (nAChRs) in the SCLC cells. The sensor was also utilized to monitor the effect of various drugs on the cells. The treatment with inhibitors such as genistin or daidzein was found to reduce Ca(2+) influx in SCLC cells. Moreover, tamoxifen, though known as the antiestrogen compound, was found to only partly block the binding of daidzein to nAChRs. Significantly, the activities of multiple individual cells could be measured repeatedly using a single sensor device, enabling statistically meaningful measurements without errors from the device-to-device variations of the sensor characteristics. This capability of the quantitative monitoring of nonadherent cells should be a major breakthrough for electrophysiology research and various biomedical applications such as drug screening and therapeutic monitoring.

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.

Nano-storage wires.

We report the development of "nano-storage wires" (NSWs), which can store chemical species and release them at a desired moment via external electrical stimuli. Here, using the electrodeposition process through an anodized aluminum oxide template, we fabricated multisegmented nanowires composed of a polypyrrole segment containing adenosine triphosphate (ATP) molecules, a ferromagnetic nickel segment, and a conductive gold segment. Upon the application of a negative bias voltage, the NSWs released ATP molecules for the control of motor protein activities. Furthermore, NSWs can be printed onto various substrates including flexible or three-dimensional structured substrates by direct writing or magnetic manipulation strategies to build versatile chemical storage devices. Since our strategy provides a means to store and release chemical species in a controlled manner, it should open up various applications such as drug delivery systems and biochips for the controlled release of chemicals.

Improved neural differentiation of human mesenchymal stem cells interfaced with carbon nanotube scaffolds.

AIM: The authors aimed to investigate the effect of carbon nanotube (CNT)-based extracellular environments on the neural differentiation of human mesenchymal stem cells (hMSCs) when combined with chemical inducers. MATERIALS & METHODS: CNT-based nanoscaffolds (linear CNT network patterns and CNT bulk network films) were prepared on solid substrates for hMSC culturing. After the hMSCs were differentiated in neural differentiation media for 2 weeks, the authors examined the neural differentiation of the hMSCs using immunocytochemistry and real-time PCR. RESULTS: The authors found that the linear CNT network patterns could effectively control the cell elongation and nuclear shape of hMSCs during the neural differentiation process, further enhancing neural gene expression compared with the bulk CNT-based films. Moreover, the CNT network films could significantly upregulate the gene expression of voltage-gated ion channels, which should be a key component for the neural activity of differentiated hMSCs. CONCLUSION: These findings suggest that CNT-based nanoscaffolds can be used as an excellent extracellular nano-/micro-environment for applications requiring effective neural differentiation of stem cells, such as regenerative medicine and tissue engineering.

Graphene-polymer hybrid nanostructure-based bioenergy storage device for real-time control of biological motor activity.

We report a graphene-polymer hybrid nanostructure-based bioenergy storage device to turn on and off biomotor activity in real-time. In this strategy, graphene was functionalized with amine groups and utilized as a transparent electrode supporting the motility of biomotors. Conducting polymer patterns doped with adenosine triphosphate (ATP) were fabricated on the graphene and utilized for the fast release of ATP by electrical stimuli through the graphene. The controlled release of biomotor fuel, ATP, allowed us to control the actin filament transportation propelled by the biomotor in real-time. This strategy should enable the integrated nanodevices for the real-time control of biological motors, which can be a significant stepping stone toward hybrid nanomechanical systems based on motor proteins.

Controlling the growth and differentiation of human mesenchymal stem cells by the arrangement of individual carbon nanotubes.

Carbon nanotube (CNT) networks on solid substrates have recently drawn attention as a means to direct the growth and differentiation of stem cells. However, it is still not clear whether cells can recognize individual CNTs with a sub-2 nm diameter, and directional nanostructured substrates such as aligned CNT networks have not been utilized to control cell behaviors. Herein, we report that human mesenchymal stem cells (hMSCs) grown on CNT networks could recognize the arrangement of individual CNTs in the CNT networks, which allowed us to control the growth direction and differentiation of the hMSCs. We achieved the directional growth of hMSCs following the alignment direction of the individual CNTs. Furthermore, hMSCs on aligned CNT networks exhibited enhanced proliferation and osteogenic differentiation compared to those on randomly oriented CNT networks. As a plausible explanation for the enhanced proliferation and osteogenic differentiation, we proposed mechanotransduction pathways triggered by high cytoskeletal tension in the aligned hMSCs. Our findings provide new insights regarding the capability of cells to recognize nanostructures smaller than proteins and indicate their potential applications for regenerative tissue engineering.