Duffy Antigen Receptor for Chemokines (DARC) Nanodisc-Based Biosensor for Detection of Staphylococcal Bicomponent Pore-Forming Leukocidins.

Staphylococcus aureus (S. aureus) is an opportunistic infectious pathogen, which causes a high mortality rate during bloodstream infections. The early detection of virulent strains in patients' blood samples is of medical interest for rapid diagnosis. The main virulent factors identified in patient isolates include leukocidins that bind to specific membrane receptors and lyse immune cells and erythrocytes. Duffy antigen receptor for chemokines (DARC) on the surface of specific cells is a main target of leukocidins such as gamma-hemolysin AB (HlgAB) and leukocidin ED (LukED). Among them, HlgAB is a conserved and critical leukocidin that binds to DARC and forms pores on the cell membranes, leading to cell lysis. Current methods are based on ELISA or bacterial culture, which takes hours to days. For detecting HlgAB with faster response and higher sensitivity, we developed a biosensor that combines single-walled carbon nanotube field effect transistors (swCNT-FETs) with immobilized DARC receptors as biosensing elements. DARC was purified from a bacterial expression system and successfully reconstituted into nanodiscs that preserve binding capability for HlgAB. Dynamic light scattering (DLS) and scanning electron microscopy (SEM) showed an increase of the DARC-containing nanodisc size in the presence of HlgAB, indicating the formation of HlgAB prepore or pore complexes. We demonstrate that this sensor can specifically detect the leukocidins HlgA and HlgAB in a quantitative manner within the dynamic range of 1 fM to 100 pM with an LOD of 0.122 fM and an LOQ of 0.441 fM. The sensor was challenged with human serum spiked with HlgAB as simulated clinical samples. After dilution for decreasing nonspecific binding, it selectively detected the toxin with a similar detection range and apparent dissociation constant as in the buffer. This biosensor was demonstrated with remarkable sensitivity to detect HlgAB rapidly and has the potential as a tool for fundamental research and clinical applications, although this sensor cannot differentiate between HlgAB and LukED as both have the same receptor.

Automated System for Attomolar-Level Detection of MiRNA as a Biomarker for Influenza A Virus.

We have developed an automated sensing system for the repeated detection of a specific microRNA (miRNA) of the influenza A (H1N1) virus. In this work, magnetic particles functionalized with DNAs, target miRNAs, and alkaline phosphate (ALP) enzymes formed sandwich structures. These particles were trapped on nickel (Ni) patterns of our sensor chip by an external magnetic field. Then, additional electrical signals from electrochemical markers generated by ALP enzymes were measured using the sensor, enabling the highly sensitive detection of target miRNA. The magnetic particles used on the sensor were easily removed by applying the opposite direction of external magnetic fields, which allowed us to repeat sensing measurements. As a proof of concept, we demonstrated the detection of miRNA-1254, one of the biomarkers for the H1N1 virus, with a high sensitivity down to 1 aM in real time. Moreover, our sensor could selectively detect the target from other miRNA samples. Importantly, our sensor chip showed reliable electrical signals even after six repeated miRNA sensing measurements. Furthermore, we achieved technical advances to utilize our sensor platform as part of an automated sensing system. In this regard, our reusable sensing platform could be utilized for versatile applications in the field of miRNA detection and basic research.

A Host Receptor Nanodisc-Based Biosensor Platform for the Detection of a Specific Virus and Its Variants.

The host receptor is a key element in the initial stage of the virus entry into the host. The use of this host receptor is valuable as a sensing element for selectively and sensitively detecting specific viruses. Also, viruses tend to escape neutralizing antibodies through viral mutation but still utilize the cell entry process using the same host receptors, so it would be a powerful detection tool even for the mutant viruses. The angiotensin-converting enzyme 2 (ACE2) receptor, which is the representative host receptor, performs an essential function in facilitating viral penetration by interacting with the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. In this study, we introduce a novel approach, where we fabricated a carbon nanotube field-effect transistor (CNT-FET) sensor and combined it with ACE2 receptor-embedded nanodisc (ND). ACE2 was produced using an E. coli expression system, purified, and integrated into the ND platform. ACE2 NDs showed robust functionality through interactions with a pseudotyped virus (PV) containing the spike protein, enabling sensitive detection of both SARS-CoV-2 and its genetic variations at 102 PFU/mL. The ACE ND-based sensor exhibited excellent selectivity by accurately differentiating SARS-CoV-2 wild-type and variants (Omicron, Delta) from other viruses (ZIKA and MERS-CoV). As a result of comparative analysis, ACE2 ND showed approximately 49% superior long-term functionality up to the second week compared to that of soluble ACE2. These findings highlight the high selectivity and sensitivity of host receptor-based sensors for detecting viral variants and provide a promising tool to prevent the spread of unknown viruses.

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.

Black Phosphorus-Based Reusable Biosensor Platforms for the Ultrasensitive Detection of Cortisol in Saliva.

A black phosphorus (BP)-based reusable biosensor platform is developed for the repeated and real-time detection of cortisol using antibody-conjugated magnetic particle (MP) structures as a refreshable receptor. Here, we took advantage of the low-noise characteristics of a mechanically exfoliated BP-based field-effect transistor (FET) and hybridized it with anti-cortisol antibody-functionalized MPs to build a highly sensitive cortisol sensor. This strategy allowed us to detect cortisol down to 1 aM in real time and discriminate cortisol from other hormones. In this case, we could easily remove MPs with used antibodies from the surface of a BP-FET and reuse the chip for up to eight repeated sensing operations. Moreover, since our platform could be fabricated using conventional photolithography techniques and the sensor can be reused multiple times, one should be able to significantly reduce operation costs for practical applications. Furthermore, this method could be utilized to detect different hormones with high sensitivity and selectivity in complex environments such as artificial saliva solutions. In this respect, our reusable BP-FET biosensing platform can be a powerful tool for versatile applications such as clinical diagnosis and basic biological analysis by conjugating various antibodies.

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.

Hydrogel-based Bioelectronic Tongue for the Evaluation of Umami Taste in Fermented Fish.

Bioelectronic tongues based on umami taste receptors have recently been reported for versatile applications such as food analyses. However, their practical applications are still limited, partly due to their limited stability and non-specific responses in real sample environments. Herein, we have developed a hydrogel-based bioelectronic tongue for the sensitive assessment of umami intensity in fish extract samples. In this study, the T1R1 venus flytrap of an umami taste receptor was immobilized on the gold floating electrodes of a carbon nanotube-based field-effect transistor. A polyacrylamide conducting hydrogel film was further hybridized on the sensor surface via physical adsorption, which could provide a good physiological environment to maintain the activity of receptors due to its excellent hydrophilicity and biocompatibility. The bioelectronic tongue with a receptor-embedded hydrogel structure showed a sensitive detection of umami substances down to 1 fM, and it also had a wide detection range of 10-15-10-2 M for monosodium glutamate and disodium inosinate, which covers the human taste threshold. More importantly, the proposed sensor could significantly reduce the non-specific binding of non-target molecules to a carbon nanotube channel as well as exhibit long-term stability, enabling sensitive detection of umami substances even in fish extract samples. Our hydrogel-based bioelectronic tongue provides a promising platform for future applications such as the flavor evaluation of foods and beverages.

Nanoscale mapping of edge-state conductivity and charge-trap activity in topological insulators.

We report the nanoscale mapping of topological edge-state conductivity and the effects of charge-traps on conductivity in a Bi2Se3 multilayer film under ambient conditions. In this strategy, we applied an electric field perpendicular to the surface plane of Bi2Se3via a conducting probe to directly map the charge-trap densities and conductivities with a nanoscale resolution. The results showed that edge regions had one-dimensional characteristics with higher conductivities (two orders) and lower charge-trap densities (four orders) than those of flat surface regions where their conductivities and charge-traps were dominated by bulk effects. Additionally, edges showed an enhanced conductivity with an elevated electric field, possibly due to the creation of new topological states by stronger spin-Hall effects. Importantly, we observed ultra-high photoconductivity predominantly on edge regions compared with that of flat surface regions, which was attributed to the excitation of edge-state carriers by light. Since our method provides an important insight into the charge transport in topological insulators, it could be a significant advancement in the development of error-tolerant topotronic devices.

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.

Adaptive biosensing platform using immune cell-based nanovesicles for food allergen detection.

Inspired by an adaptive immune system, we have developed a bioelectronic sensing platform which relies on nanovesicles for a signal amplification and can be easily adapted for the detection of new food allergens. In this work, nanovesicles with anti-immunoglobulin E (anti-IgE) antibody receptors were extracted from immune cells and immobilized on a carbon nanotube-based transistor to build a highly sensitive and selective biosensing platform. Our sensor could detect peanut allergen, arachis hypogaea 2 (Ara h 2), down to 0.1 fM and selectively discriminate target allergens in real food samples such as peanut and egg white. As a proof of concept, we demonstrated the detection of different target molecules using the same nanovesicles linked with different antibodies. Our sensor platform was also utilized to quantitatively evaluate the effect of allergy drug such as cromolyn. In this regard, our strategy can be utilized for basic research and versatile applications in food and pharmacological industries.

Bioelectronic Tongues Mimicking Insect Taste Systems for Real-Time Discrimination between Natural and Artificial Sweeteners.

A bioelectronic tongue (B-ET) mimicking insect taste systems is developed for the real-time detection and discrimination of natural and artificial sweeteners. Here, a carbon nanotube field-effect transistor (CNT-FET) was hybridized with nanovesicles including the honeybee sugar taste receptor, gustatory receptor 1 of Apis mellifera (AmGr1). This strategy allowed us to detect glucose, a major component of nectar, down to 100 fM in real time and identify sweet tastants from other tastants. It could also be utilized for the detection of glucose in dextrose tablet solutions. Importantly, we demonstrated the discrimination between natural and artificial sweeteners down to 10 pM even in real beverages such as decaffeinated coffee using our hybrid platform. In this respect, our B-ET mimicking insect taste systems can be a powerful tool for various applications such as food screening and basic studies on insect taste systems.

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.

Nanoscale Mapping of Carrier Mobilities in the Ballistic Transports of Carbon Nanotube Networks.

Much progress has been made in the nanoscale analysis of nanostructures, while the mapping of key charge transport properties such as a carrier mobility remains a challenge, especially for one-dimensional systems. Here, we report the nanoscale mapping of carrier mobilities in carbon nanotube (CNT) networks and show that charge transport behaviors varied depending on network structures. In this work, the spatial distribution of localized charge transport properties such as mobilities and charge trap densities in CNT networks were mapped via a scanning noise microscopy. The mobility map was obtained from the conductivity maps measured at different back-gate biases, showing up to two orders of mobility variations depending on localized network structures. Furthermore, from the maps, correlations between mobility/conductivity and charge trap density were analyzed to determine charge transport mechanisms. In metallic CNT networks, the regions with rather high (low) or low (high) charge trap densities (mobilities) exhibited a diffusive or ballistic transport behavior, respectively. Interestingly, semiconducting CNT networks also exhibited a gradual transition from a diffusive to a ballistic transport behavior as the CNT mobility was increased by reaching the on-state with negative gate biases. The mapping of the cross-patterned CNT network showed that metallic CNT electrodes could achieve a good electrical contact with semiconducting CNTs without high contact resistance regions. Since this method allowed one to map versatile charge transport properties such as mobility, conductivity, and charge trap density, it can be a powerful tool for basic research about charge transport phenomena and practical device applications.

Dipolar Noise in Fluorinated Molecular Wires.

We demonstrate a strategy to directly map and quantify the effects of dipole formation on electrical transports and noises in the self-assembled monolayers (SAMs) of molecular wires. In this method, the SAM patterns of fluorinated molecules with dipole moments were prepared on conducting substrates, and a conducting probe in contact-mode atomic force microscopy was utilized to map currents and noises through the probe on the molecular patterns. The maps were analyzed to extract the characteristic parameters of dipolar noises in SAMs, and the results were compared with those of hydrogenated molecular patterns without dipole moments. At rather low bias conditions, the fluorinated molecular junctions exhibited a tunneling conduction and a resistance value comparable to that of the hydrogenated molecules with a six-times-longer length, which was attributed to stronger dipoles formation in fluorinated molecules. Interestingly, conductance (G) in different regions of fluorinated molecular patterns exhibited a strong correlation with a noise power spectral density of SI/I2 like SI/I2 ∝ G-2, which can be explained by enhanced barrier fluctuations produced by the dipoles of fluorinated molecules. Furthermore, we observed that the noise power spectral density of fluorinated molecules showed an anomalous frequency (f) dependence like SI/I2 ∝ 1/f1.7, possibly due to the slowing down of the tunneling of carriers from increased barrier fluctuations. In rather high bias conditions, conductions in both hydrogenated and fluorinated molecules showed a transition from tunneling to thermionic charge transports. Our results provide important insights into the effects of dipoles on mesoscopic transport and resistance-fluctuation in molecules and could have a significant impact on the fundamental understanding and applications in this area.

Ultrasensitive Bioelectronic Tongue Based on the Venus Flytrap Domain of a Human Sweet Taste Receptor.

Sweet taste is an important factor that regulates calorie intake and contributes to food preferences in humans and animals. Therefore, the evaluation of sweet substances is essential for various fields such as healthcare, food, and pharmaceutical industries. Sweet tastants are detected by sweet taste receptors which are class C G-protein-coupled receptors. T1R2 venus flytrap (VFT) of the sweet taste receptor is known as a primary ligand-binding domain for sweet tastants. In this study, we developed an ultrasensitive artificial sweet taste bioelectronic tongue based on the T1R2 VFT of a human sweet taste receptor. Here, the T1R2 VFT of a human sweet taste receptor was successfully overexpressed in a bacterial expression system. A T1R2 VFT-immobilized carbon nanotube field-effect transistor with floating electrodes was exploited as an artificial sweet taste sensory system. Significantly, our T1R2 VFT-functionalized bioelectronic tongue could be used to detect solutions of sweet tastants down to 0.1 fM and selectively discriminate sweet substances from other taste substances. Furthermore, our device could be used to monitor the response of the T1R2 VFT domain of a sweet taste receptor to sweet substances in real food environments such as apple juice and chamomile herb tea. Moreover, our device was used to evaluate the inhibition and enhancement effects on sweet taste receptors by zinc ions and chamomile tea, respectively. In addition, our device demonstrated long-term storability and reusability. In this respect, our sweet taste bioelectronic tongue could be a promising tool for various basic research and industrial applications.

Evaluation of site-selective drug effects on GABA receptors using nanovesicle-carbon nanotube hybrid devices.

Site-selective drug effects on the ion-channel activities of γ-aminobutyric acid type A (GABAA) receptors are evaluated by using a nanovesicle-carbon nanotube hybrid device. Here, nanovesicles containing GABAA receptors are immobilized on the channel region of a carbon nanotube field-effect transistor. The receptor responses of this hybrid device to GABA are detected with a high sensitivity down to ∼1 aM even in the presence of other neurotransmitters. Further, sensitivity differences between two GABAA-receptor-subunit compositions of α5ß2γ2 and α1ß2γ2 are assessed by normalizing the dose-dependent responses obtained from these hybrid devices. Specifically, the GABA concentration that produces 50% of maximal response (EC50) is obtained as ∼10 pM for α5ß2γ2 subunits and ∼1 nM for α1ß2γ2 subunits of GABAA receptor. Significantly, the potency profiles of both antagonist and agonist of GABAA receptor can be evaluated by analyzing EC50 values in the presence and absence of those drugs. A competitive antagonist increases the EC50 value of GABA by binding to the same site as GABA, while an allosteric agonist reduces it by binding to a different site. These results indicate that this hybrid device can be a powerful tool for the evaluation of candidate drug substances modulating GABA-mediated neurotransmission.

Mapping the nanoscale effects of charge traps on electrical transport in grain structures of indium tin oxide thin films.

We report the mapping of the nanoscale effects of charge trap activities in the grain structures of an oxygen plasma-treated indium tin oxide (ITO) thin film. Here, a conducting Pt probe made direct contact with the surface of an ITO thin film and scanned the surface while measuring the maps of electrical currents and noises. The measured data were analyzed to obtain the maps of sheet conductance (G s) and charge trap density (N eff) in the grain structures of the ITO thin film. The results showed that grain boundaries exhibited a lower sheet conductance and a higher charge trap density than those of the regions inside grains. Interestingly, the scaling behavior of G s ∝ N eff -0.5 was observed in both grain and boundary regions, indicating diffusive charge transport. Furthermore, the sheet conductance increased by two times, and the density of charge traps decreased by ∼70% after an oxygen plasma treatment, presumably due to the enhanced crystallinity of the ITO film. Interestingly, in some boundary regions, the sheet conductance and the charge trap density exhibited the scaling behavior of G s ∝ N eff 0.5, which was attributed to the hopping conduction caused by the enhanced crystallinity and increased localized states in the boundary regions. Since our method provides valuable insights into charge transport and charge trap activities in transparent conducting thin films, it can be a powerful tool for basic research and practical optoelectronic device applications based on ITO thin films.

Reusable surface amplified nanobiosensor for the sub PFU/mL level detection of airborne virus.

We developed a reusable surface-amplified nanobiosensor for monitoring airborne viruses with a sub-PFU/mL level detection limit. Here, sandwich structures consisted of magnetic particles functionalized with antibodies, target viruses, and alkaline phosphatases (ALPs) were formed, and they were magnetically concentrated on Ni patterns near an electrochemical sensor transducer. Then, the electrical signals from electrochemical markers generated by ALPs were measured with the sensor transducer, enabling highly-sensitive virus detection. The sandwich structures in the used sensor chip could be removed by applying an external magnetic field, and we could reuse the sensor transducer chip. As a proof of concepts, the repeated detection of airborne influenza virus using a single sensor chip was demonstrated with a detection limit down to a sub-PFU/mL level. Using a single reusable sensor transducer chip, the hemagglutinin (HA) of influenza A (H1N1) virus with different concentrations were measured down to 10 aM level. Importantly, our sensor chip exhibited reliable sensing signals even after more than 18 times of the repeated HA sensing measurements. Furthermore, airborne influenza viruses collected from the air could be measured down to 0.01 PFU/mL level. Interestingly, the detailed quantitative analysis of the measurement results revealed the degradation of HA proteins on the viruses after the air exposure. Considering the ultrasensitivity and reusability of our sensors, it can provide a powerful tool to help preventing epidemics by airborne pathogens in the future.

Reusable surface plasmon resonance biosensor chip for the detection of H1N1 influenza virus.

We developed a reusable magnetic surface plasmon resonance (SPR) sensor chip for detecting various target molecules repeatedly in a conventional SPR system. Here, ferromagnetic patterns on a SPR sensor chip were utilized to trap a layer of magnetic particles, and they were utilized as a solid substrate for SPR sensing in a conventional SPR system. After a sensing experiment, the used magnetic particles were removed by external magnetic fields, and a new layer of magnetic particles was immobilized to the SPR sensor chip for additional sensing measurements. Since magnetic particles were trapped on the ferromagnetic patterns, we could use our reusable SPR chip for SPR sensing measurements in a traditional SPR system without any applied magnetic fields. Significantly, ferromagnetic patterns on the sensor chip surface deflected the strong external fields, so that the large aggregation of magnetic particles on the sensor surface was reduced. We demonstrated using a single reusable SPR sensor chip to measure the nucleoprotein (NP) of H1N1 influenza virus solution ranging repeatedly for more than 7 times without significant signal degradation. Also, different target molecules could be repeatedly measured in a single SPR chip. Since our reusable SPR sensor chip can be repeatedly used in a conventional SPR system without any chemical processes for refreshment, the cost for SPR sensing should be significantly reduced. In this case, our reusable SPR sensor chip can be a major breakthrough and can be used for versatile practical applications of SPR sensors.