Urinary dengue NS1 detection on Au-decorated ZnO nanowire platform.

Biodetection for non-invasive diagnostics of fluids, especially urine, remains a challenge to scientists due to low target concentrations. And biological complexes of the detection target may contain contaminants that also interfere with any assay. Dengue non-structural 1 protein (Dengue NS1) is an important biomarker for dengue hemorrhagic fever and dengue shock syndrome. Here, we developed an Au-decorated nanowire platform and applied it with a sandwich fluorophore-linked immunosorbent well plate assay (FLISA) to detect Dengue NS1 in urine. For the platform, we fabricated zinc oxide (ZnO) nanowires to provide a high surface area and then coated them with gold nanoparticles (ZnO/Au nanowires) to simply modify the Dengue NS1 antibody and enhance the fluorescence intensity. Our platform employs a sandwich FLISA that exhibits high sensitivity, specifically detecting Dengue NS1 with a limit of detection (LOD) of 1.35 pg/mL. This LOD was 4500-fold lower than the LOD of a commercially available kit for Dengue NS1 enzyme-linked immunosorbent assay. We believe that our ZnO/Au nanowire platform has the potential to revolutionize the field of non-invasive diagnostics for dengue.

Resistive Pulse Sensing on a Capillary-Assisted Microfluidic Platform for On-Site Single-Particle Analyses.

Capillary-assisted flow is valuable for utilizing microfluidics-based electrical sensing platforms at on-site locations by simplifying microfluidic operations and system construction; however, incorporating capillary-assisted flow in platforms requires easy microfluidic modification and stability over time for capillary-assisted flow generation and sensing performance. Herein, we report a capillary-assisted microfluidics-based electrical sensing platform using a one-step modification of polydimethylsiloxane (PDMS) with polyethylene glycol (PEG). As a model of electrical sensing platforms, this work focused on resistive pulse sensing (RPS) using a micropore in a microfluidic chip for label-free electrical detection of single analytes, and filling the micropore with an electrolyte is the first step to perform this RPS. The PEG-PDMS surfaces remained hydrophilic after ambient storage for 30 d and assisted in generating an electrolyte flow for filling the micropore with the electrolyte. We demonstrated the successful detection and size analysis of micrometer particles and bacterial cells based on RPS using the microfluidic chip stored in a dry state for 30 d. Combining this capillary-assisted microfluidic platform with a portable RPS system makes on-site detection and analysis of single pathogens possible.

Metal Oxide Nanostructures Enhanced Microfluidic Platform for Efficient and Sensitive Immunofluorescence Detection of Dengue Virus.

Rapid and sensitive detection of Dengue virus remains a critical challenge in global public health. This study presents the development and evaluation of a Zinc Oxide nanorod (ZnO NR)-surface-integrated microfluidic platform for the early detection of Dengue virus. Utilizing a seed-assisted hydrothermal synthesis method, high-purity ZnO NRs were synthesized, characterized by their hexagonal wurtzite structure and a high surface-to-volume ratio, offering abundant binding sites for bioconjugation. Further, a comparative analysis demonstrated that the ZnO NR substrate outperformed traditional bare glass substrates in functionalization efficiency with 4G2 monoclonal antibody (mAb). Subsequent optimization of the functionalization process identified 4% (3-Glycidyloxypropyl)trimethoxysilane (GPTMS) as the most effective surface modifier. The integration of this substrate within a herringbone-structured microfluidic platform resulted in a robust device for immunofluorescence detection of DENV-3. The limit of detection (LOD) for DENV-3 was observed to be as low as 3.1 × 10-4 ng/mL, highlighting the remarkable sensitivity of the ZnO NR-integrated microfluidic device. This study emphasizes the potential of ZnO NRs and the developed microfluidic platform for the early detection of DENV-3, with possible expansion to other biological targets, hence paving the way for enhanced public health responses and improved disease management strategies.

Spatial exosome analysis using cellulose nanofiber sheets reveals the location heterogeneity of extracellular vesicles.

Extracellular vesicles (EVs), including exosomes, are recognized as promising functional targets involved in disease mechanisms. However, the intravital heterogeneity of EVs remains unclear, and the general limitation for analyzing EVs is the need for a certain volume of biofluids. Here, we present cellulose nanofiber (CNF) sheets to resolve these issues. We show that CNF sheets capture and preserve EVs from ~10 µL of biofluid and enable the analysis of bioactive molecules inside EVs. By attaching CNF sheets to moistened organs, we collect EVs in trace amounts of ascites, which is sufficient to perform small RNA sequence analyses. In an ovarian cancer mouse model, we demonstrate that CNF sheets enable the detection of cancer-associated miRNAs from the very early phase when mice did not have apparent ascites, and that EVs from different locations have unique miRNA profiles. By performing CNF sheet analyses in patients, we identify further location-based differences in EV miRNA profiles, with profiles reflecting disease conditions. We conduct spatial exosome analyses using CNF sheets to reveal that ascites EVs from cancer patients exhibit location-dependent heterogeneity. This technique could provide insights into EV biology and suggests a clinical strategy contributing to cancer diagnosis, staging evaluation, and therapy planning.

Engineering Interface Defects and Interdiffusion at the Degenerate Conductive In2O3/Al2O3 Interface for Stable Electrodes in a Saline Solution.

A low-temperature Al2O3 deposition process provides a simplified method to form a conductive two-dimensional electron gas (2DEG) at the metal oxide/Al2O3 heterointerface. However, the impact of key factors of the interface defects and cation interdiffusion on the interface is still not well understood. Furthermore, there is still a blank space in terms of applications that go beyond the understanding of the interface's electrical conductivity. In this work, we carried out a systematic experimental study by oxygen plasma pretreatment and thermal annealing post-treatment to study the impact of interface defects and cation interdiffusion at the In2O3/Al2O3 interface on the electrical conductance, respectively. Combining the trends in electrical conductance with the structural characteristics, we found that building a sharp interface with a high concentration of interface defects provides a reliable approach to producing such a conductive interface. After applying this conductive interface as electrodes for fabricating a field-effect transistor (FET) device, we found that this interface electrode exhibited ultrastability in phosphate-buffered saline (PBS), a commonly used biological saline solution. This study provides new insights into the formation of conductive 2DEGs at metal oxide/Al2O3 interfaces and lays the foundation for further applications as electrodes in bioelectronic devices.

Identifying high-grade serous ovarian carcinoma-specific extracellular vesicles by polyketone-coated nanowires.

Cancer cell-derived extracellular vesicles (EVs) have unique protein profiles, making them promising targets as disease biomarkers. High-grade serous ovarian carcinoma (HGSOC) is the deadly subtype of epithelial ovarian cancer, and we aimed to identify HGSOC-specific membrane proteins. Small EVs (sEVs) and medium/large EVs (m/lEVs) from cell lines or patient serum and ascites were analyzed by LC-MS/MS, revealing that both EV subtypes had unique proteomic characteristics. Multivalidation steps identified FRα, Claudin-3, and TACSTD2 as HGSOC-specific sEV proteins, but m/lEV-associated candidates were not identified. In addition, for using a simple-to-use microfluidic device for EV isolation, polyketone-coated nanowires (pNWs) were developed, which efficiently purify sEVs from biofluids. Multiplexed array assays of sEVs isolated by pNW showed specific detectability in cancer patients and predicted clinical status. In summary, the HGSOC-specific marker detection by pNW are a promising platform as clinical biomarkers, and these insights provide detailed proteomic aspects of diverse EVs in HGSOC patients.

Mutation detection of urinary cell-free DNA via catch-and-release isolation on nanowires for liquid biopsy.

Cell-free DNA (cfDNA) and extracellular vesicles (EVs) are molecular biomarkers in liquid biopsies that can be applied for cancer detection, which are known to carry information on the necessary conditions for oncogenesis and cancer cell-specific activities after oncogenesis, respectively. Analyses for both cfDNA and EVs from the same body fluid can provide insights into screening and identifying the molecular subtypes of cancer; however, a major bottleneck is the lack of efficient and standardized techniques for the isolation of cfDNA and EVs from clinical specimens. Here, we achieved catch-and-release isolation by hydrogen bond-mediated binding of cfDNA in urine to zinc oxide (ZnO) nanowires, which also capture EVs by surface charge, and subsequently we identified genetic mutations in urinary cfDNA. The binding strength of hydrogen bonds between single-crystal ZnO nanowires and DNA was found to be equal to or larger than that of conventional hydrophobic interactions, suggesting the possibility of isolating trace amounts of cfDNA. Our results demonstrated that nanowire-based cancer screening assay can screen cancer and can identify the molecular subtypes of cancer in urine from brain tumor patients through EV analysis and cfDNA mutation analysis. We anticipate our method to be a starting point for more sophisticated diagnostic models of cancer screening and identification.

All-in-One Nanowire Assay System for Capture and Analysis of Extracellular Vesicles from an ex Vivo Brain Tumor Model.

Extracellular vesicles (EVs) have promising potential as biomarkers for early cancer diagnosis. The EVs have been widely studied as biological cargo containing essential biological information not only from inside vesicles such as nucleic acids and proteins but also from outside vesicles such as membrane proteins and glycolipids. Although various methods have been developed to isolate EVs with high yields such as captures based on density, size, and immunoaffinity, different measurement systems are needed to analyze EVs after isolation, and a platform that enables all-in-one analysis of EVs from capture to detection in multiple samples is desired. Since a nanowire-based approach has shown an effective capability for capturing EVs via surface charge interaction compared to other conventional methods, here, we upgraded the conventional well plate assay to an all-in-one nanowire-integrated well plate assay system (i.e., a nanowire assay system) that enables charge-based EV capture and EV analysis of membrane proteins. We applied the nanowire assay system to analyze EVs from brain tumor organoids in which tumor environments, including vascular formations, were reconstructed, and we found that the membrane protein expression ratio of CD31/CD63 was 1.42-fold higher in the tumor organoid-derived EVs with a p-value less than 0.05. Furthermore, this ratio for urine samples from glioblastoma patients was 2.25-fold higher than that from noncancer subjects with a p-value less than 0.05 as well. Our results demonstrated that the conventional well plate method integrated with the nanowire-based EV capture approach allows users not only to capture EVs effectively but also to analyze them in one assay system. We anticipate that the all-in-one nanowire assay system will be a powerful tool for elucidating EV-mediated tumor-microenvironment crosstalk.

Non-competitive fluorescence polarization immunosensing for CD9 detection using a peptide as a tracer.

This paper is the first report of a non-competitive fluorescence polarization immunoassay (NC-FPIA) using a peptide as a tracer. The NC-FPIA can easily and quickly quantify the target after simply mixing them together. This feature is desirable for point-of-need applications such as clinical diagnostics, infectious disease screening, on-site analysis for food safety, etc. In this study, the NC-FPIA was applied to detect CD9, which is one of the exosome markers. We succeeded in detecting not only CD9 but also CD9 expressing exosomes derived from HeLa cells. This method can be applied to various targets if a tracer for the target can be prepared, and expectations are high for its future uses.

Breath odor-based individual authentication by an artificial olfactory sensor system and machine learning.

Breath odor sensing-based individual authentication was conducted for the first time using an artificial olfactory sensor system. Using a 16-channel chemiresistive sensor array and machine learning, a mean accuracy of >97% was successfully achieved. The impact of the number of sensors on the accuracy and reproducibility was also demonstrated.

Correction: Breath odor-based individual authentication by an artificial olfactory sensor system and machine learning.

Correction for 'Breath odor-based individual authentication by an artificial olfactory sensor system and machine learning' by Chaiyanut Jirayupat et al., Chem. Commun., 2022, DOI: https://doi.org/10.1039/D1CC06384G.

Tailoring ZnO nanowire crystallinity and morphology for label-free capturing of extracellular vesicles.

Zinc oxide (ZnO) nanowires have shown their potential in isolation of cancer-related biomolecules such as extracellular vesicles (EVs), RNAs, and DNAs for early diagnosis and therapeutic development of diseases. Since the function of inorganic nanowires changes depending on their morphology, previous studies have established strategies to control the morphology and have demonstrated attainment of improved properties for gas and organic compound detection, and for dye-sensitized solar cells and photoelectric conversion performance. Nevertheless, crystallinity and morphology of ZnO nanowires for capturing EVs, an important biomarker of cancer, have not yet been discussed. Here, we fabricated ZnO nanowires with different crystallinities and morphologies using an ammonia-assisted hydrothermal method, and we comprehensively analyzed the crystalline nature and oriented growth of the synthesized nanowires by X-ray diffraction and selected area electron diffraction using high resolution transmission electron microscopy. In evaluating the performance of label-free EV capture in a microfluidic device platform, we found both the crystallinity and morphology of ZnO nanowires affected EV capture efficiency. In particular, the zinc blende phase was identified as important for crystallinity, while increasing the nanowire density in the array was important for morphology to improve EV capture performance. These results highlighted that the key physicochemical properties of the ZnO nanowires were related to the EV capture performance.

Water-Selective Nanostructured Dehumidifiers for Molecular Sensing Spaces.

Humidity and moisture effects, frequently called water poisoning, in surroundings are inevitable for various molecular sensing devices, strongly affecting their sensing characteristics. Here, we demonstrate a water-selective nanostructured dehumidifier composed of ZnO/TiO2/CaCl2 core-shell heterostructured nanowires for molecular sensing spaces. The fabricated nanostructured dehumidifier is highly water-selective without detrimental adsorptions of various volatile organic compound molecules and can be repeatedly operated. The thermally controllable and reversible dehydration process of CaCl2·nH2O thin nanolayers on hydrophilic ZnO/TiO2 nanowire surfaces plays a vital role in such water-selective and repeatable dehumidifying operations. Furthermore, the limitation of detection for sensing acetone and nonanal molecules in the presence of moisture (relative humidity ∼ 90%) was improved more than 20 times using nanocomposite sensors by operating the developed nanostructured dehumidifier. Thus, the proposed water-selective nanostructured dehumidifier offers a rational strategy and platform to overcome water poisoning issues for various molecular and gas sensors.

Oxide nanowire microfluidics addressing previously-unattainable analytical methods for biomolecules towards liquid biopsy.

Nanowire microfluidics using a combination of self-assembly and nanofabrication technologies is expected to be applied to various fields due to its unique properties. We have been working on the fabrication of nanowire microfluidic devices and the development of analytical methods for biomolecules using the unique phenomena generated by the devices. The results of our research are not just limited to the development of nanospace control with "targeted dimensions" in "targeted arrangements" with "targeted materials/surfaces" in "targeted spatial locations/structures" in microfluidic channels, but also cover a wide range of analytical methods for biomolecules (extraction, separation/isolation, and detection) that are impossible to achieve with conventional technologies. Specifically, we are working on the extraction technology "the cancer-related microRNA extraction method in urine," the separation technology "the ultrafast and non-equilibrium separation method for biomolecules," and the detection technology "the highly sensitive electrical measurement method." These research studies are not just limited to the development of biomolecule analysis technology using nanotechnology, but are also opening up a new academic field in analytical chemistry that may lead to the discovery of new pretreatment, separation, and detection principles.

Molecular profiling of extracellular vesicles via charge-based capture using oxide nanowire microfluidics.

Extracellular vesicles (EVs) have shown promising features as biomarkers for early cancer diagnoses. The outer layer of cancer cell-derived EVs consists of organotropic metastasis-induced membrane proteins and specifically enriched proteoglycans, and these molecular compositions determine EV surface charge. Although many efforts have been devoted to investigating the correlation between EV subsets obtained through density-, size-, and immunoaffinity-based captures and expressed membrane proteins, understanding the correlation between EV subsets obtained through surface charge-based capture and expressed membrane proteins is lacking. Here, we propose a methodology to profile membrane proteins of EV subsets obtained through surface charge-based capture. Nanowire-induced charge-based capture of EVs and in-situ profiling of EV membrane proteins are the two key methodology points. The oxide nanowires allowed EVs to be obtained through surface charge-based capture due to the diverse isoelectric points of the oxides and the large surface-to-volume ratios of the nanowire structures. And, with the ZnO nanowire device, whose use does not require any purification and concentration processes, we demonstrated the correlation between negatively-charged EV subsets and expressed membrane proteins derived from each cell. Furthermore, we determined that a colon cancer related membrane protein was overexpressed on negatively charged surface EVs derived from colon cancer cells.

Annealed ZnO/Al2O3 Core-Shell Nanowire as a Platform to Capture RNA in Blood Plasma.

RNA analytical platforms gained extensive attention recently for RNA-based molecular analysis. However, the major challenge for analyzing RNAs is their low concentration in blood plasma samples, hindering the use of RNAs for diagnostics. Platforms that can enrich RNAs are essential to enhance molecular detection. Here, we developed the annealed ZnO/Al2O3 core-shell nanowire device as a platform to capture RNAs. We showed that the annealed ZnO/Al2O3 core-shell nanowire could capture RNAs with high efficiency compared to that of other circulating nucleic acids, including genomic DNA (gDNA) and cell-free DNA (cfDNA). Moreover, the nanowire was considered to be biocompatible with blood plasma samples due to the crystalline structure of the Al2O3 shell which serves as a protective layer to prevent nanowire degradation. Our developed device has the potential to be a platform for RNA-based extraction and detection.

Fabrication of a Robust In2O3 Nanolines FET Device as a Biosensor Platform.

Field-effect transistors (FETs) are attractive biosensor platforms for rapid and accurate detection of various analytes through surface immobilization of specific bio-receptors. Since it is difficult to maintain the electrical stability of semiconductors of sensing channel under physiological conditions for long periods, passivation by a stable metal oxide dielectric layer, such as Al2O3 or HfO2, is currently used as a common method to prevent damage. However, protecting the sensing channel by passivation has the disadvantage that the distance between the target and the conductive channel increases, and the sensing signal will be degraded by Debye shielding. Even though many efforts use semiconductor materials directly as channels for biosensors, the electrical stability of semiconductors in the physiological environments has rarely been studied. In this work, an In2O3 nanolines FET device with high robustness in artificial physiological solution of phosphate buffered saline (PBS) was fabricated and used as a platform for biosensors without employing passivation on the sensing channel. The FET device demonstrated reproducibility with an average threshold voltage (VTH) of 5.235 V and a standard deviation (SD) of 0.382 V. We tested the robustness of the In2O3 nanolines FET device in PBS solution and found that the device had a long-term electrical stability in PBS with more than 9 days' exposure. Finally, we demonstrated its applicability as a biosensor platform by testing the biosensing performance towards miR-21 targets after immobilizing the phosphonic acid terminated DNA probes. Since the surface immobilization of multiple bioreceptors is feasible, we demonstrate that the robust In2O3 FET device can be an excellent biosensor platform for biosensors.

The impact of surface Cu2+ of ZnO/(Cu1-x Zn x )O heterostructured nanowires on the adsorption and chemical transformation of carbonyl compounds.

The surface cation composition of nanoscale metal oxides critically determines the properties of various functional chemical processes including inhomogeneous catalysts and molecular sensors. Here we employ a gradual modulation of cation composition on a ZnO/(Cu1-x Zn x )O heterostructured nanowire surface to study the effect of surface cation composition (Cu/Zn) on the adsorption and chemical transformation behaviors of volatile carbonyl compounds (nonanal: biomarker). Controlling cation diffusion at the ZnO(core)/CuO(shell) nanowire interface allows us to continuously manipulate the surface Cu/Zn ratio of ZnO/(Cu1-x Zn x )O heterostructured nanowires, while keeping the nanowire morphology. We found that surface exposed copper significantly suppresses the adsorption of nonanal, which is not consistent with our initial expectation since the Lewis acidity of Cu2+ is strong enough and comparable to that of Zn2+. In addition, an increase of the Cu/Zn ratio on the nanowire surface suppresses the aldol condensation reaction of nonanal. Surface spectroscopic analysis and theoretical simulations reveal that the nonanal molecules adsorbed at surface Cu2+ sites are not activated, and a coordination-saturated in-plane square geometry of surface Cu2+ is responsible for the observed weak molecular adsorption behaviors. This inactive surface Cu2+ well explains the mechanism of suppressed surface aldol condensation reactions by preventing the neighboring of activated nonanal molecules. We apply this tailored cation composition surface for electrical molecular sensing of nonanal and successfully demonstrate the improvements of durability and recovery time as a consequence of controlled surface molecular behaviors.

Rapid Discrimination of Extracellular Vesicles by Shape Distribution Analysis.

A rapid and simple cancer detection method independent of cancer type is an important technology for cancer diagnosis. Although the expression profiles of biological molecules contained in cancer cell-derived extracellular vesicles (EVs) are considered candidates for discrimination indexes to identify any cancerous cells in the body, it takes a certain amount of time to examine these expression profiles. Here, we report the shape distributions of EVs suspended in a solution and the potential of these distributions as a discrimination index to discriminate cancer cells. Distribution analysis is achieved by low-aspect-ratio nanopore devices that enable us to rapidly analyze EV shapes individually in solution, and the present results reveal a dependence of EV shape distribution on the type of cells (cultured liver, breast, and colorectal cancer cells and cultured normal breast cells) secreting EVs. The findings in this study provide realizability and experimental basis for a simple method to discriminate several types of cancerous cells based on rapid analyses of EV shape distributions.

Urinary MicroRNA-Based Diagnostic Model for Central Nervous System Tumors Using Nanowire Scaffolds.

There are no accurate mass screening methods for early detection of central nervous system (CNS) tumors. Recently, liquid biopsy has received a lot of attention for less-invasive cancer screening. Unlike other cancers, CNS tumors require efforts to find biomarkers due to the blood-brain barrier, which restricts molecular exchange between the parenchyma and blood. Additionally, because a satisfactory way to collect urinary biomarkers is lacking, urine-based liquid biopsy has not been fully investigated despite the fact that it has some advantages compared to blood or cerebrospinal fluid-based biopsy. Here, we have developed a mass-producible and sterilizable nanowire-based device that can extract urinary microRNAs efficiently. Urinary microRNAs from patients with CNS tumors (n = 119) and noncancer individuals (n = 100) were analyzed using a microarray to yield comprehensive microRNA expression profiles. To clarify the origin of urinary microRNAs of patients with CNS tumors, glioblastoma organoids were generated. Glioblastoma organoid-derived differentially expressed microRNAs (DEMs) included 73.4% of the DEMs in urine of patients with parental tumors but included only 3.9% of those in urine of noncancer individuals, which suggested that many CNS tumor-derived microRNAs could be identified in urine directly. We constructed the diagnostic model based on the expression of the selected microRNAs and found that it was able to differentiate patients and noncancer individuals at a sensitivity and specificity of 100 and 97%, respectively, in an independent dataset. Our findings demonstrate that urinary microRNAs extracted with the nanowire device offer a well-fitted strategy for mass screening of CNS tumors.