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A novel strategy for epidermal growth factor receptor (EGFR) detection using a cell-based field-effect transistor (FET) with enzymatic chemical signal amplification is proposed. Four human breast cancer cell lines [BT474, MDA-MB-231 (MM231), MDA-MB-468 (MM468), and MDA-MB-453 (MM453)] were used to compare the expression levels of EGFR. The cells were non-specifically captured on the surface of the gate of the FET, irrespective of their surface antigens. With this configuration, the heterogeneity of the cells would be analyzed using secondary antibodies conjugated to different kinds of enzymes. Four breast cancer cell lines with different levels of EGFR expression were captured on the respective surfaces of the extracellular matrix (ECM) gel-coated gates of the FETs. Glucose oxidase (GOx) was conjugated to the secondary antibody, and the output signals of the cell-based FETs changed depending on the expression levels of EGFR upon addition of glucose. The order of the expression levels of EGFR among the four cell lines, determined with the cell-based FETs, was consistent with the results of fluorescence detection determined by fluorescence-activated cell sorting (FACS). The cell-based FETs are advantageous for miniaturization and in massive parallel analyses of target molecules expressed on the membranes of cells and EVs, and their small size and cost effectiveness for cancer testing could enable their realization in a future liquid biopsy.

Resistive pulse sensing (RPS) is an analytical method that can be used to individually count particles from a small sample. RPS simply monitors the physical characteristics of particles, such as size, shape, and charge density, and the integration of RPS with biosensing is an attractive theme to detect biological particles such as virus and bacteria. In this report, a methodology of biosensing on RPS was investigated. Polydopamine (PD), an adhesive component of mussels, was used as the base material to create a sensing surface. PD adheres to most materials, such as noble metals, metal oxides, semiconductors, and polymers; as a result, PD is a versatile intermediate layer for the fabrication of a biosensing surface. As an example of a biological particle, human influenza A virus (H1N1 subtype) was used to monitor translocation of particles through the pore membrane. When virus-specific ligands (6'-sialyllactose) were immobilized on the pore surface, the translocation time of the virus particles was considerably extended. The detailed translocation data suggest that the viral particles were trapped on the sensing surface by specific interactions. In addition, virus translocation processes on different pore surfaces were distinguished using machine learning. The result shows that the simple and versatile PD-based biosensor surface design was effective. This advanced RPS measurement system could be a promising analytical technique.

Epithelial barriers that seal cell gaps by forming tight junctions to prevent the free permeation of nutrients, electrolytes, and drugs, are essential for maintaining homeostasis in multicellular organisms. The development of nanocarriers that can permeate epithelial tissues without compromising barrier function is key for establishing a safe and efficient drug delivery system (DDS). Previously, we have demonstrated that a water-soluble phospholipid-mimicking random copolymer, poly(2-methacryloyloxyethyl phosphorylcholine30-random-n-butyl methacrylate70) (PMB30W), enters the cytoplasm of live cells by passive diffusion mechanisms, without damaging the cell membranes. The internalization mechanism was confirmed to be amphiphilicity-induced membrane fusion. In the present study, we demonstrated nonendocytic permeation of PMB30W through the model epithelial barriers of Madin-Darby canine kidney (MDCK) cell monolayers in vitro. The polymer penetrated epithelial MDCK monolayers via transcellular pathways without breaching the barrier functions. This was confirmed by our unique assay that can monitor the leakage of the proton as the smallest indicator across the epithelial barriers. Moreover, nonendocytic transepithelial permeation was achieved when insulin was chemically conjugated with the phospholipid-mimicking nanocarrier. The bioactivity of insulin as a growth factor was found to be maintained even after translocation. These fundamental findings may aid the establishment of transepithelial DDS with advanced drug efficiency and safety.

The capture and detection of cells expressing a breast-cancer related membrane protein, namely a BT474 cell line expressing HER2, is demonstrated using ion-sensitive field effect transistors (ISFETs). BT474 cells were exposed to anti-HER2 antibodies and urease-conjugated secondary antibodies to induce chemical signal amplification by adding urea.

A solid-state potentiometric biosensor based on the organic and inorganic mixed phase modification of a silver surface is proposed. Stabilization of the electromotive force and functionalization with biomolecules on the sensing surface were simultaneously achieved using silver chloride chemically deposited with 1,3-diaminopropanetetraacetic acid ferric ammonium salt monohydrate and a self-assembled monolayer with oligonucleotide probes, respectively. The formation of silver chloride and adsorption of alkanethiol on the silver surface were confirmed with X-ray photoelectron spectroscopy. The resulting modified surface reduced the nonspecific binding of interfering biomolecules and achieved a high signal to noise ratio. The electromotive forces of the modified silver thin film electrodes were stable under constant chloride ion concentrations. Hybridization assays were performed to detect microRNA 146. The lower limit of detection was 0.1 pM because of the small standard deviation. The proposed biosensor could be useful as a disposable single-use sensor in medical fields such as liquid biopsies.

Endosomal escape is crucial for the delivery of nucleic acids. However, the understanding of the underlying mechanisms is still deficient. In this work, we explored the effects of lipid- and polymer-based transfection reagents on the permeability of cellular membranes through an innovative method combining a proton-sensing transistor and a cytosolic LDH leakage assay, which allows us to distinguish between modes of molecule permeation that may occur during endosomal escape. By testing the commercial reagents lipofectin and in vivo JetPEI under physiological and endosomal pH conditions, we found that both lipid- and polymer-based transfection reagents have pH-dependent pore-forming activity, with the former creating smaller pores than the latter. This versatile approach of assessing carrier-membrane interactions is expected to contribute to the development of next-generation nucleic acid delivery systems.

Researchers widely apply enzyme inhibition to chemicals such as pesticides, nerve gases, and anti-Alzheimer's drugs. However, application of enzyme inhibition to odorant sensors is less common because the corresponding reaction mechanisms have not yet been clarified in detail. In this study, we propose a new strategy for highly selective detection of odorant molecules by using an inhibitor-specific enzyme. As an example, we analyzed the selective interactions between acetylcholinesterase (AChE) and limonene─the major odorant of citrus and an AChE inhibitor─using molecular dynamics simulations. In these simulations, limonene was found to be captured at specific binding sites of AChE by modifying the binding site of acetylcholine (ACh), which induced inhibition of the catalytic activity of AChE toward ACh hydrolysis. We confirmed the simulation results by experiments using an ion-sensitive field-effect transistor, and the degree of inhibition of ACh hydrolysis depended on the limonene concentration. Accordingly, we quantitatively detected limonene at a detection limit of 5.7 μM. We furthermore distinguished the response signals to limonene from those to other odorants, such as pinene and perillic acid. Researchers will use our proposed odorant detection method for other odorant-enzyme combinations and applications of miniaturized odorant-sensing systems based on rapid testing.

Detection and isolation of sialic acid (SA) over-expressed biomolecules is of continuous interest in cancer-related research. Boronic acid-based SA-recognition chemistry provides a synthetic molecu...

Understanding the interactions of soft nanomatters with cell membranes is particularly important for research into nanocarrier-based drug delivery systems, cell engineering, and subcellular imaging. Most nanoparticles, vesicles, micelles, and polymeric aggregates are internalized into endosomes and, eventually, lysosomes in the cytosol because of energy-dependent endocytic processes. Endocytic uptake substantially limits the access to the cytoplasm where a cargo agent acts. Bypassing the endocytic pathways by direct penetration into plasma membrane barriers would enhance the efficacy of nanomedicines. Some zwitterionic polymer nanoaggregates have been shown to permeate into the cell interior in an energy-independent manner. We have elucidated this phenomenon by observing changes in the biomembrane barrier functions against protons as the smallest indicator and have used these results to further design and develop poly(betaines). In this work, we investigated the translocation mechanisms for a series of zwitterionic poly(methacrylamide) and poly(methacrylate) species bearing a pyridinium propane sulfonate moiety in the monomers. Minor differences in the monomer structures and functional groups were observed to have dramatic effects on the interaction with plasma membranes during translocation. The ability to cross the plasma membrane involves a balance among the betaine dipole-dipole interaction, NH-π interaction, π-π interaction, cation-π interaction, and amide hydrogen bonding. We found that the cell-penetrating polysulfobetaines had limited or no detrimental effect on cell proliferation. Our findings enhance the opportunity to design and synthesize soft nanomatters for cell manipulations by passing across biomembrane partitions.

Accumulating evidence demonstrates that not only sustained elevation of blood glucose levels but also the glucose fluctuation represents key determinants for diabetic complications and mortality. Current closed-loop insulin therapy option is limited to the use of electronics-based systems, although it poses some technical issues with high cost. Here we demonstrate an electronics-free, synthetic boronate gel-based insulin-diffusion-control device technology that can cope with glucose fluctuations and potentially address the electronics-derived issues. The gel was combined with hemodialysis hollow fibers and scaled suitable for rats, serving as a subcutaneously implantable, insulin-diffusion-active site in a manner dependent on the subcutaneous glucose. Continuous glucose monitoring tests revealed that our device not only normalizes average glucose level of rats, but also markedly ameliorates the fluctuations over timescale of a day without inducing hypoglycemia. With inherent stability, diffusion-dependent scalability, and week-long & acute glucose-responsiveness, our technology may offer a low-cost alternative to current electronics-based approaches., Here, the authors develop an in vivo insulin delivery system which consists of a glucose responsive gel combined with hemodialysis hollow fibers. This system is electronics-free, temperature independent, and can stably sustain acute glucose-responsiveness in rats.

Insulin delivery in a self-regulated and painless way to tightly control the glycemic level is highly demanded for diabetes treatment. Phenylboronic acid (PBA) has gained great research interests d...

Eradication of cancer stem cells (CSCs) is an ultimate goal in cancer chemotherapy. Although a ligand-assisted targeting approach seems rational, the existence of subpopulations of CSCs and their discrimination from those present on healthy sites makes it a severe challenge. Some boronic acid (BA) derivatives are known for the ability to bind with glycan-terminal sialic acid (SA), in a manner dependent on the acidification found in hypoxic tumoral microenvironment. Taking advantage of this feature, here we show that the BA-ligand fluorescence conjugate can effectively target multiple CSC subpopulations in parallel, which otherwise must be independently aimed when using antibody--ligands.

Recently, phenylboronic acid (PBA) gel containing microneedle (MN) technology with acute and sustained glucose-sensitive functionality has attracted significant research attention. Herein, we report a polyvinyl alcohol(PVA)-coated MNs patch with an interconnected porous gel drug reservoir for enhanced skin penetration efficiency and mechanical strength. The hybrid MNs patch fabricated with a novel, efficient method displayed a “cake-like” two-layer structure, with the tip part being composed of boronate-containing smart gel attached to a porous gel layer as a drug reservoir. The porous structure provides the necessary structural support for skin insertion and space for insulin loading. The mechanical strength of the hybrid MNs patch was further enhanced by surface coating with crystallized PVA. Compared with MNs patches attached to hollow drug reservoirs, this hybrid MNs patch with a porous gel reservoir was shown to be able to penetrate the skin more effectively, and is promising for on-demand, long-acting transdermal insulin delivery with increased patient compliance.

Phosphorylcholine (PC) has been used to improve the water solubility and biocompatibility of biomaterials. Here, we show that PC can also work as a ligand for targeting cancer cells based on their increased phospholipid metabolism. PC-installed multiarm poly(ethylene glycol)s and polymeric micelles achieved high and rapid internalization in pancreatic cancer cells. This enhanced cellular uptake was drastically reduced when the cells were incubated with excess free PC or at 4 °C, as well as by inhibiting the phospholipid transfer protein (PLTP) on the surface of cancer cells, indicating an energy dependent active transport mediated by PLTP.

Periodontal disease is an inflammatory disorder that is triggered by bacterial plaque and causes the destruction of the tooth-supporting tissues leading to tooth loss. Several bacteria species, including Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, are considered to be associated with severe periodontal conditions. In this study, we demonstrated a quartz crystal microbalance (QCM) immunoassay for quantitative assessment of the periodontal bacteria, A. actinomycetemcomitans. An immunosensor was constructed using a self-assembled monolayer of 11-mercaptoundecanoic acid (11-MUA) on the gold surface of a QCM chip. The 11-MUA layer was evaluated using a cyclic voltammetry technique to determine its mass and packing density. Next, a monoclonal antibody was covalently linked to 11-MUA using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide to act as the biorecognition element. The specificity of the monoclonal antibody was confirmed by an enzyme-linked immunosorbent assay. A calibration curve, for the relationship between the frequency shifts and number of bacteria, was used to calculate the number of A. actinomycetemcomitans bacteria in a test sample. Based on a regression equation, the lower detection limit was 800 cells, with a dynamic range up to 2.32 × 106 cells. Thus, the QCM biosensor in this study provides a sensitive and label-free method for quantitative analysis of periodontal bacteria. The method can be used in various biosensing assays for practical application and routine detection of periodontitis pathogens.

In the fields of tissue engineering and drug discovery, confirming the formation and maturation of epithelial cell tight junctions (TJs), which are necessary for blocking pathogenic invasion and absorption of nutrients and ions, at a high spatiotemporal resolution is essential. We previously developed a system of monitoring pH perturbation induced by weak acid exposure to cells cultured on an ion-sensitive field-effect transistor that enables a sensitive and specific detection of biomembrane injuries and TJ breakdowns caused by external stimuli such as nanomaterials and cytotoxins. In this study, we monitor time-lapse changes in the paracellular diffusion of growing epithelial cell monolayers using the pH perturbation assay as well as conventional permeability and trans-epithelial electrical resistance assays. The effects of the extracellular matrix and a TJ potentiator (KN-93) on epithelial TJ formation are evaluated. TJ formations were promoted on the substrate coated with Matrigel more than on the one coated with poly(l-lysine). KN-93 accelerated TJ formations in a dose-dependent manner. The pH perturbation assay denoted a longer incubation time for the completion of TJ formation compared with the conventional assays under the same conditions. Importantly, the pH perturbation assay is able to rigorously evaluate TJ formation, as the assay uses protons as the smallest indicator for detecting paracellular gaps, and the pH perturbation is specific to TJ alterations. These features for in vitro TJ evaluation using proton dynamics are advantageous for applications in tissue engineering and drug development.

A redox-sensitive inter-conversion between ascorbic acid (ASC) and its oxidized form dehydroascorbic acid (DHA) in the intracellular environment has been of exceptional interest to recent metabolomics and pharmaceutical research. We developed a chromatographic protocol to instantly determine these vitamers with each identity from cellular extracts, without any labeling and pretreatments. Owing to its simplicity, one can readily continue the assay for hours, an otherwise difficult to cover timescale at which the intracellular DHA-ASC conversion comes into play. The method was validated for the analysis of pancreatic cancer cells, to our knowledge the first-ever study on a nucleated cell type, to trace in detail their kinetics of glucose transporter-dependent DHA uptake and, simultaneously, that for the intracellular ASC conversion. The simplest of all the relevant techniques and yet with the unique ability to provide each vitamer identity on a high-throughput basis, this method should offer the most practical option for VC-involved physiological and pharmaceutical studies including high-dose VC cancer therapy.

We propose an artificial intelligence-based chemical-sensing system integrating a porous gate field-effect transistor (PGFET) array modified by gas chromatography stationary phase materials and machine-learning techniques. The chemically sensitive PGFET array generates cross-reactive signals for computational analysis and shows potential for applications to compact intelligent sensing devices, including mobile electronic noses.

Tight junctions (TJs) in the epithelial cell gap play primary roles in body defense and nutrient absorption in multicellular organisms. Standard in vitro assays lack sensitivity, selectivity, temporal resolution, and throughput for bioengineering applications. Prompted by the rigorous barrier functions of TJ, we developed a TJ assay that senses proton leaks in the cell gap using ion-sensitive field-effect transistors. Upon exposure of Madin-Darby canine kidney (MDCK) cells cultured on a gate dielectric to a calcium-chelator EGTA, ammonia-assisted pH perturbation was enhanced solely in TJ-forming cells. This was supported by simulations with increased ion permeability in the paracellular pathway. Following administration of Clostridium perfringens enterotoxin as a specific TJ inhibitor to the MDCK cells, our method detected TJ breakdown at a 13× lower concentration than a conventional trans-epithelial electrical resistance assay. Thus, the semiconductor-based active pH sensing could offer an alternative to current in vitro assays for TJs in pathological, toxicological, and pharmaceutical studies.

In this paper, we report the demonstration of odorant capture and detection with a high-sensitivity olfactory system having a membrane-type odorant capture module. We carried out the odorant capture experiment with limonene and skatole gases as representative odorants, and the captured concentration was 7.6 nM and 0.2 µM, respectively. Furthermore, we have demonstrated 10 nM sensitivity by the graphene FET biosensor, which implies that our olfactory sensing system can realize from gas capture to sensing.

Graphene’s remarkable attributes make it suitable for application to biosensors for biomolecular recognition. Specific and precise target detection is realized by designing robust methods for immobilization of probe molecules, such as oligonucleotides, antibodies, receptors, and sugar chains, to a device surface. In this research, we developed a chemical modification method with a plasma treatment of amino groups on natural defects of graphene, which is compatible with a wafer-scalable semiconductor process, to prevent deterioration of the carrier mobility. The plasma treatment was optimized in terms of the efficiency of the amino radical generation, length of the mean free path, and reaction energy on graphene. The density of the modified amino groups on graphene was approximately 0.065 groups/nm², and the change in the ΔId/ΔVg characteristic of the graphene field-effect transistor (FET) was negligible. DNA probes were then attached to the amino groups on the graphene FET. The target complementary DNA was detected at 1 nM after hybridization using the graphene FET devices. The plasma-assisted modification of the amino groups on the graphene surface was developed for immobilization of the DNA probes, and hybridization with the target DNA was demonstrated without deterioration of the carrier mobility.

Despite tremendous complexity in glycan structure, sialic acid (SA) provides an analytically accessible index for glycosylation, owing to its uniquely anionic nature and glycan-chain terminal occupation. Taking advantage of boronic acid (BA) based SA-recognition chemistry, we here demonstrate a label-free, no enzymatic, potentiometric determination of fetuin, a blood-circulating glycoprotein implicated in physiological and various pathological states. A phenylboronic acid (PBA) ω-end-functionalized poly(ethylene glycol) (PEG) with an α-tethering unit bearing pendent alkyne groups was "grafted-to" a gold electrode modified with 11-azide-undecathiol by a copper-catalyzed azide-alkyne cycloaddition reaction. Using the electrode, fetuin was potentiometrically detectable with a μM-order-sensitivity that is comparable to what is found in blood-collected specimen. Our finding may have implications for developing a remarkably economic hemodiagnostic technology with ease of downsizing and mass production.

Microneedle (MN) technology, which can transdermally deliver insulin in a noninvasive manner, offers a promising way to replace subcutaneous self-injection for diabetes management. Hydrogel is an attractive candidate for MN fabrication because of its biocompatibility, controllable degradability, and possibility to achieve sustained as well as stimuli-responsive drug delivery. Herein, we report a smart MN composed of a semi-interpenetrating network (semi-IPN) hydrogel prepared by biocompatible silk fibroin (SF) and phenylboronic acid/acrylamide for glucose-responsive insulin delivery. Six fabrication methods were investigated to maintain the glucose sensitivity of the hydrogel while avoiding deformation during fabrication. The optimized method was to fabricate smart MNs using a two-layer strategy, with a needle region formed by the SF combined semi-IPN hydrogel and the base layer fabricated by SF. The hybrid MN autonomously released insulin well-correspondent to the glucose change pattern via the regulation of the skin layer formed on the surface. Furthermore, this hybrid MN retained its original needle shape after 1 week in aqueous system, thus eliminating the safety concerns associated with dissolving MNs and suggesting the possibility for sustained delivery. This nondegradable smart MN is promising to provide on-demand insulin in a long-acting, painless, and convenient way.

mRNA is a promising therapeutic nucleic acid, although effective delivery systems are required for its broad application. Polyion complex (PIC) micelles loading mRNA via polyion complexation with block catiomers are emerging as promising carriers for mRNA delivery, but the PIC stability has been limited so far. Controlling the binding of polycations to mRNA could affect the micelle stability. Nevertheless, the impact of binding affinity between polycations and mRNA on the function of mRNA-loaded PIC micelles (mRNA/m) remains unknown. Herein, we review our recent orthogonal approaches controlling the stiffness and the valency of polycations to improve the performance of mRNA/m toward enhancing stability and delivery efficiency. Thus, block catiomers with contrasting flexibility were developed to prepare mRNA/m. The flexible catiomer stabilized mRNA/m against enzymatic attack and polyanion exchange compared to the rigid catiomer, promoting protein translation in vitro and in vivo, and prolonged mRNA bioavailability in blood after systemic injection. Based on these observations, we also developed flexible catiomers with different valencies. The guanidinated catiomer stabilized mRNA/m compared to the aminated catiomers, facilitating intracellular delivery and eventual gene expression. Our findings indicate the importance of controlling the polymer binding to mRNA for developing flexible polycation-based systems directed to in vivo applications.

Tracing cell surface sialylation dynamics at a scale of the glycolipoprotein microdomain (lipid rafts) formations remains an intriguing challenge of cellular biology. Here, we demonstrate that this goal is accessible, taking advantage of a boronic acid (BA)-based reversible molecular recognition chemistry. A BA-end-functionalized poly(ethylene glycol) was decorated onto an atomic force microscopy (AFM) cantilever, which provided a dynamic and sialic acid (SA)-specific imaging mode. Using this technique, we were able to heat map the SA expression levels not only on protein-decorated substrates but also directly on the cell surfaces, with a submicrometer scale resolution that may be relevant to that of the lipid rafts formation. The SA specificity and the binding reversibility of the probe were confirmed from its pH-dependent characteristics and an inhibition assay using free state SA. This finding may provide a noninvasive means for assessing a variety of SA-involved glycosylation dynamics spanning from physiology to pathology.

Chemical and enzymatic modifications of amino acid residues in protein after translation contain rich information about physiological conditions and diseases. Histone acetylation/deacetylation is the essential post-translational modification by regulating gene transcription. Such qualitative changes of biomacromolecules need to be detected in point-of-care systems for an early and accurate diagnosis. However, there is no technique to aid this issue. Previously, we have applied an aptamer-functionalized field-effect transistor (FET) to the specific protein biosensing. Quantitative changes of target protein in a physiological solution have been determined by detecting innate charges of captured protein at the gate-solution interface. Moreover, we have succeeded in developing an integrated system of FET and quartz crystal microbalance (QCM) sensors for determining the adsorbed mass and charge, simultaneously or in parallel. Prompted by this, in this study, we developed a new label-free method for detecting histone acetylation using FET and QCM sensors. The loss of positive charge of lysine residue by chemically induced acetylation of histone subunits (H3 and H4) was successfully detected by potentiometric signals using anti-histone aptamer-functionalized FET. The adsorbed mass was determined by the same anti-histone aptamer-functionalized QCM. From these results, the degree of acetylation was correlated to the charge-to-mass ratio of histone subunits. The histone required for the detection was below 100 nM, owing to the high sensitivity of aptamer-functionalized FET and QCM sensors. These findings will guide us to a new way of measuring post-translational modification of protein in a decentralized manner for an early and accurate diagnosis.

Understanding the interactions of eukaryotic cellular membranes with nanomaterials is required to construct efficient and safe nanomedicines and molecular bioengineering. Intracellular uptake of nanocarriers by active endocytosis limits the intracellular distribution to the endosomal compartment, impairing the intended biological actions of the cargo molecules. Nonendocytic intracellular migration is another route for nanomaterials with cationic or amphiphilic properties to evade the barrier function of the lipid bilayer plasma membranes. Direct transport of nanomaterials into cells is efficient, but this may cause cytotoxic or biocidal effects by temporarily disrupting the biological membrane barrier. We have recently discovered that nonendocytic internalization of synthetic amphipathic polymer-based nanoaggregates that mimic the structure of natural phospholipids can occur without inducing cytotoxicity. Analysis using a proton leakage assay indicated that the polymer enters cells by amphiphilicity-induced membrane fusion rather than by transmembrane pore formation. These noncytotoxic cell-penetrating polymers may find applications in drug delivery systems, gene transfection, cell therapies, and biomolecular engineering.

Significant progress in the field of solid-state biosensors has occurred over the last decade. Various types of sensing devices with high-density integration and flexible configuration, as well as new applications for clinical diagnosis and healthcare, have been developed using blood, serum, and other body fluids such as sweat, tears, and saliva. In this review, we focus on the recent progress of solid-state biosensors in the biomedical field. New concepts, technical achievements, and practical applications of the sensors are described and discussed from the perspective of sensing materials, sensor structures, design of biomolecular recognition, and biomedical applications. Several challenges remain before the solid-state biosensors are realized for point-of-care testing in clinical diagnosis and healthcare. Collaboration among researchers, integration of technologies, and knowledge from different fields are necessary to tackle the remaining challenges.

Resistive pulse sensing (RPS) is an analytical technique for detecting particles with nano- to micrometer diameters, such as proteins, viruses, and bacteria. RPS is a promising tool for diagnosis as it can analyze the characteristics of target particles individually from ion current blockades as pulse waveforms. However, it is difficult to discriminate analog targets because RPS merely provides physical information such as size, shape, concentration, and charge density of the analyte. Influenza A virus, which is 80-120 nm in diameter, has various subtypes, demonstrating the diversity of virus characteristics. For example, highly pathogenic avian influenza infections in humans are recognized as an emerging infectious disease with high mortality rates compared with human influenza viruses. Distinguishing human from avian influenza using their differing biological characteristics would be challenging using RPS. To develop a highly selective diagnostic system for infectious diseases, we combined RPS with molecular recognition. Gold nanoparticles (GNPs) that have human influenza A (H1N1 subtype) virus-specific sialic acid receptors on the surface were prepared as a virus label for RPS analysis. A sulfobetaine and sialic acid (ligand) hybrid surface was formed on the GNPs for the suppression of nonspecific interaction. The results show a size change of viruses derived from specific interactions with GNPs. In contrast, no size shift was observed when nonspecific sialic acid receptor-immobilized GNPs were used. Detection of viruses by individual particle counting could be a new facet of diagnosis.

An organic electrochemical transistor (OECT) with a trisaccharide-grafted conductive polymer channel was developed for human influenza A virus detection under aqueous conditions. A target recognition element was introduced into the electrochemical amplifier of the OECT for highly sensitive, selective, and label-free virus sensing. 3,4-Ethylenedioxythiophene (EDOT) and its derivative bearing an oxylamine moiety (EDOTOA) were electrochemically copolymerized on the channel region composed of a PEDOT:PSS thin film. The trisaccharides composed of Sia-α2,6′-Gal-Glu (2,6-sialyllactose), a specific receptor for the hemagglutinin used as a spike protein on the surface of human influenza A virus, were covalently introduced into the EDOTOA unit. Changes in the drain current of the OECT were observed following virus adsorption onto the 2,6-sialyllactose-functionalized channel. A signal transduction mechanism involving a doping effect due to the adsorption of negatively-charged virus nanoparticles is proposed. The limit of detection was more than two orders of magnitude lower than commercial immunochromatographic influenza virus assays over the same detection time. Because of its processability with printing technologies and low power consumption, the OECT device developed here may be suitable for the wearable monitoring of influenza virus infection.

A quantitative diagnostic method for dental caries would improve oral health, which directly affects the quality of life. Here we describe the preparation and application of Ir/IrOx pH sensors, which are used to measure the surface pH of dental caries. The pH level is used as an indicator to distinguish between active and arrested caries. After a dentist visually inspected and defined 18 extracted dentinal caries at various positions as active or arrested caries, the surface pH values of sound and caries areas were directly measured with an Ir/IrOx pH sensor with a diameter of 300 μm as a dental explorer. The average pH values of the sound root, the arrested caries, and active caries were 6.85, 6.07, and 5.30, respectively. The pH obtained with an Ir/IrOx sensor was highly correlated with the inspection results by the dentist, indicating that the types of caries were successfully categorized. This caries testing technique using a micro Ir/IrOx pH sensor provides an accurate quantitative caries evaluation and has potential in clinical diagnosis.

Molecular recognition is one of the key factors in designing biosensors due to which nanowires functionalized with molecular recognition have attracted a lot of attention as promising candidates for nanostructures embedded in biosensors. However, the difficulty in real-world applications with analytical specificity is that molecular recognition on nanowires mainly depends on antibody modification with multistep modification procedures. Furthermore, the antibody modification suffers from nonspecific adsorption of undesired proteins in body fluid on the nanowires, which causes false responses and lowers sensitivity. Herein, we propose biomolecular recognition using surface-modified nanowires via thiolated 2-methacryloxyethyl phosphorylcholine (MPC-SH). MPC-SH enables self-assembled monolayer (SAM) modification, which contributes to the reduction of nonspecific adsorption of biomolecules onto the nanowires, and the specific capture of a target protein is attained in the presence of calcium ions. Our concept demonstrates the recognition of the biomarker protein on nanowire surfaces modified by MPC-SH SAM with a single step modification procedure.

Conductive polymers, such as poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS), can be used for specific biosensing after its surface is modified with functional groups. Herein, we report a simple two-step surface modification method for commercially available PEDOT:PSS films printed on a flexible substrate. The derivatization process involves: (1) the introduction of an ethylenediamine linker for conjugation with the biorecognition element by activating the sulfonate group in PSS with an acid chloride reaction; and (2) the introduction of FPBA into the ethylenediamine linker. As a proof of concept, 4-carboxy-3-fluorophenylboronic acid (carboxy FPBA) is covalently introduced to the side chain of the PSS dopant in the conductive layer. FPBA serves as a selective molecular recognition element for diol compounds, including glucose, at neutral pH. Continuous glucose monitoring is achieved by label-free potentiometry using FPBA-functionalized commercial PEDOT:PSS films. Negative potential responses with glucose are successfully achieved in ideal and realistic buffer solutions with or without 1% fetal bovine serum (FBS) because of the complexation of FPBA. The dynamic range of the enzyme-free sensor covered physiologically relevant blood glucose levels of 70–140 mg/dL. The developed method is suitable and cost effective for the mass production of biosensors as it can be applied to a large surface area at once. Furthermore, various ligands and receptors can be introduced onto commercial PEDOT:PSS films using this technique. Hence, the proposed method has potential applications in wearable and implantable biosensors.