Ionizable Polymeric Micelles with Phenylalanine Moieties Enhance Intracellular Delivery of Self-Replicating RNA for Long-Lasting Protein Expression In Vivo.

mRNA-based therapeutics are revolutionizing the landscape of medical interventions. However, the short half-life of mRNA and transient protein expression often limits its therapeutic potential, demanding high treatment doses or repeated administrations. Self-replicating RNA (RepRNA)-based treatments could offer enhanced protein production and reduce the required dosage. Here, we developed polymeric micelles based on flexible poly(ethylene glycol)-poly(glycerol) (PEG-PG) block copolymers modified with phenylalanine (Phe) moieties via biodegradable ester bonds for the efficient delivery of RepRNA. These polymers successfully encapsulated RepRNA into sub-100 nm micelles assisted by the hydrophobicity of the Phe moieties and their ability to π-π stack with the bases in RepRNA. The micelles made from Phe-modified PEG-PG (PEG-PG(Phe)) effectively maintained the integrity of the loaded RepRNA in RNase-rich serum conditions. Once taken up by cells, the micelles triggered a pH-responsive membrane disruption, promoted by the strong protonation of the amino groups at endosomal pH, thereby delivering the RepRNA to the cytosol. The system induced strong protein expression in vitro and outperformed commercial transfecting reagents in vivo, where it resulted in enhanced and long-lasting protein expression.

Sialic acid biosensing by post-printing modification of PEDOT:PSS with pyridylboronic acid.

A poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS)-based conducting polymer, which has biorecognition capabilities, has promising biosensing applications. Previously, we developed a facile method for post-printing chemical modification of PEDOT:PSS thin films from commercial sources. Molecular recognition elements were directly introduced into the PSS side chain by a two-step chemical reaction: introduction of an ethylenediamine linker via an acid chloride reaction of the sulfonate moiety, and subsequent receptor attachment to the linker via amine coupling. In this study, the same method was used to introduce 6-carboxypyridine-3-boronic acid (carboxy-PyBA) into the linker for specifically detecting N-acetylneuraminic acid (sialic acid, SA), as a cancer biomarker. The surface-modified PEDOT:PSS films were characterized by X-ray photoelectron spectroscopy, attenuated total reflection Fourier-transform infrared spectroscopy, and static water contact angle and conductivity measurements. The specific interaction between PyBA and SA was detected by label-free reagent-free potentiometry. The SA-specific negative potential responses of modified PEDOT:PSS electrodes, which was ascribed to an SA carboxyl anion, were observed in a physiologically relevant SA range (1.6-2.9 mM) at pH 5, in a concentration-dependent manner even in the presence of 10% fetal bovine serum. The sensitivity was -2.9 mV/mM in 1-5 mM SA with a limit of detection of 0.7 mM. The sensing performances were almost equivalent to those of existing graphene-based electrical SA sensors. These results show that our chemical derivatization method for printing PEDOT:PSS thin films will have applications in SA clinical diagnostics.

Transepithelial delivery of insulin conjugated with phospholipid-mimicking polymers via biomembrane fusion-mediated transcellular pathways.

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-n­butyl methacrylate70) (PMB30W), enters the cytoplasm of live cells by passive diffusion manners, without damaging the cell membranes. The internalization mechanism was confirmed to be amphiphilicity-induced membrane fusion. In the present study, we demonstrated energy-independent 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, energy-independent 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. STATEMENT OF SIGNIFICANCE: A nanocarrier that can freely permeate epithelial tissues without compromising barrier function is key for successful DDS. Existing strategies mainly rely on paracellular transport associated with tight junction breakdown or transcellular transport via transporter recognition-mediated active uptake. These approaches raise concerns about efficiency and safety. In this study, we performed non-endocytic permeation of phospholipid-mimicking polymers through the model epithelial barriers in vitro. The polymer penetrated via transcytotic pathways without breaching the barriers of biomembrane and tight junction. Moreover, transepithelial permeation occurred when insulin was covalently attached to the nanocarrier. The bioactivity of insulin was maintained even after translocation. The biomimetic design of nanocarrier may realize safe and efficient transepithelial DDS.

Chemically Induced pH Perturbations for Analyzing Biological Barriers Using Ion-Sensitive Field-Effect Transistors.

Potentiometric pH measurements have long been used for the bioanalysis of biofluids, tissues, and cells. A glass pH electrode and ion-sensitive field-effect transistor (ISFET) can measure the time course of pH changes in a microenvironment as a result of physiological and biological activities. However, the signal interpretation of passive pH sensing is difficult because many biological activities influence the spatiotemporal distribution of pH in the microenvironment. Moreover, time course measurement suffers from stability because of gradual drifts in signaling. To address these issues, an active method of pH sensing was developed for the analysis of the cell barrier in vitro. The microenvironmental pH is temporarily perturbed by introducing a low concentration of weak acid (NH4+) or base (CH3COO-) to cells cultured on the gate insulator of ISFET using a superfusion system. Considering the pH perturbation originates from the semi-permeability of lipid bilayer plasma membranes, induced proton dynamics are used for analyzing the biomembrane barriers against ions and hydrated species following interaction with exogenous reagents. The unique feature of the method is the sensitivity to the formation of transmembrane pores as small as a proton (H+), enabling the analysis of cell-nanomaterial interactions at the molecular level. The new modality of cell analysis using ISFET is expected to be applied to nanomedicine, drug screening, and tissue engineering.

A proton/macromolecule-sensing approach distinguishes changes in biological membrane permeability during polymer/lipid-based nucleic acid delivery.

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.

Organic and inorganic mixed phase modification of a silver surface for functionalization with biomolecules and stabilization of electromotive force.

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.

Label-Free Monitoring of Histone Acetylation Using Aptamer-Functionalized Field-Effect Transistor and Quartz Crystal Microbalance Sensors.

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.

Internalization Mechanisms of Pyridinium Sulfobetaine Polymers Evaluated by Induced Protic Perturbations on Cell Surfaces.

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.

Phospholipid-mimicking cell-penetrating polymers: principles and applications.

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.

Poly(3,4-ethylenedioxythiophene) Bearing Pyridylboronic Acid Group for Specific Recognition of Sialic Acid.

Conducting polymers tethered with molecular recognition elements are good candidates for biosensing applications such as detecting a target molecule with selectivity. We develop a new monomer, namely, 3,4-ethylenedioxythiophene bearing a pyridylboronic acid moiety (EDOT-PyBA), for label-free detection of sialic acid as a cancer biomarker. PyBA, which is known to show specific binding to sialic acid in acid conditions is used as a synthetic ligand instead of lectins. PyBA confirms the enhanced binding affinity for sialic acid at pH 5.0-6.0 compared with traditional phenylboronic acid. Poly(EDOT-PyBA) is electrodeposited on a planar glassy carbon electrode and the obtained film is successfully characterized by X-ray photoelectron spectroscopy, scanning electron microscopy, atomic force microscopy, water contact angle measurements, and electrochemical impedance spectroscopy. The specific interaction of PyBA with sialic acid at the solution/electrode interface is detected by differential pulse voltammetry in a dynamic range 0.1-3.0 mM with a detection limit of 0.1 mM for a detection time of 3 min. The sensitivity covers the total level of free sialic acid in human serum and the assay time is the shorter than that of other methods. The poly(EDOT-PyBA) electrode successfully detects spiked sialic acid in human serum samples. Owing to its processability, mass productivity, and robustness, polythiophene conjugated with "boronolectin" is a candidate material for developing point-of-care and wearable biosensors.

Induced Proton Dynamics on Semiconductor Surfaces for Sensing Tight Junction Formation Enhanced by an Extracellular Matrix and Drug.

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.

Translocation Mechanisms of Cell-Penetrating Polymers Identified by Induced Proton Dynamics.

Unlike the majority of nanomaterials designed for cellular uptake via endocytic pathways, some of the functional nanoparticles and nanospheres directly enter the cytoplasm without overt biomembrane injuries. Previously, we have shown that a water-soluble nanoaggregate composed of amphiphilic random copolymer of 2-methacryloyloxyethyl phosphorylcholine (MPC) and n-butyl methacrylate (BMA), poly(MPC- random-BMA) (PMB), passes live cell membranes in an endocytosis-free manner. Yet, details in its translocation mechanism remain elusive due to the lack of proper analytical methods. To understand this phenomenon experimentally, we elaborated the original pH perturbation assay that is extremely sensitive to the pore formation on cell membranes. The ultimate sensitivity originates from the detection of the smallest indicator H+ (H3O+) passed through the molecularly sized transmembrane pores upon challenge by exogenous reagents. We revealed that water-soluble PMB at the 30 mol % MPC unit (i.e., PMB30W) penetrated into the cytosol of model mammalian cells without any proton leaks, in contrast to conventional cell-penetrating peptides, TAT and R8 as well as the surfactant, Triton X-100. While exposure of PMB30W permeabilized cytoplasmic lactate dehydrogenase out of the cells, indicating the alteration of cell membrane polarity by partitioning of amphiphilic PMB30W into the lipid bilayers. Nevertheless, the biomembrane alterations by PMB30W did not exhibit cytotoxicity. In summary, elucidating translocation mechanisms by proton dynamics will guide the design of nanomaterials with controlled permeabilization to cell membranes for bioengineering applications.

Induced Proton Perturbation for Sensitive and Selective Detection of Tight Junction Breakdown.

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.

Gold Nanoparticles with Ligand/Zwitterion Hybrid Layer for Individual Counting of Influenza A H1N1 Subtype Using Resistive Pulse Sensing.

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.

Electrodeposition of Zwitterionic PEDOT Films for Conducting and Antifouling Surfaces.

Conferring antifouling properties can extend the use of conducting polymers in biosensors and bioelectronics under complex biological conditions. On the basis of the antifouling properties of a series of zwitterionic polymers, we synthesized new thiophene-based compounds bearing a phosphorylcholine, carboxybetaine, or sulfobetaine pendant group. The monomers were synthesized by a facile reaction of thiol-functionalized 3,4-ethylenedioxythiophene with zwitterionic methacrylates. Electrochemical copolymerization was performed to deposit zwitterionic poly(3,4-ethylenedioxythiophene) (PEDOT) films with tunable conducting and antifouling properties on a conducting substrate. Electrochemical impedance spectroscopy showed that the conductivity and capacitance decreased with increasing zwitterionic content in the films. Protein adsorption and cell adhesion studies showed the effects of the type and content of zwitterions on the antifouling characteristics. Optimization of the electrodeposition conditions enabled development of both conducting and antifouling polymer films. These antifouling conjugated functional polymers have promising applications in biological environments.

Biomolecular recognition on nanowire surfaces modified by the self-assembled monolayer.

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.

pH Mapping on Tooth Surfaces for Quantitative Caries Diagnosis Using Micro Ir/IrOx pH Sensor.

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.

Specific binding of human C-reactive protein towards supported monolayers of binary and engineered phospholipids.

Circulating C-reactive protein (CRP) recognizes altered plasma membranes and activates complements systems in the acute phase of inflammation and infection in human. We have shown previously the calcium-independent adsorption of CRP toward 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and lysophosphatidylcholine (LPC) on supported phospholipid monolayers. Here, we extended our study to other phospholipids and additives to elucidate the pattern recognition of CRP using a surface plasmon resonance biosensor. Surface density and lateral fluidity depended on the type of phospholipids in the monolayers as characterized by SPR and fluorescence recovery after photobleaching measurements. CRP recognized 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG) in the supported POPC monolayers without calcium at pH 7.4 and 5.5. As opposed to LPC, CRP did not recognize 3-sn-lysophosphatidylethanolamine in the POPC monolayers in calcium-free conditions. While, the addition of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) or sphingomyelin to supported POPC monolayers blocked CRP adsorption. Calcium-dependent CRP binding was observed only at pH 5.5 on supported monolayers of engineered phospholipids with inverted headgroups relative to POPC. The complement 1q (C1q) protein recognized the active form of CRP on the supported phospholipid monolayers. The discovery of CRP recognition with these phospholipids aids our understanding of the activation dynamics of CRP with phospholipid-based biomaterials when used during the acute phase.

Identification of types of membrane injuries and cell death using whole cell-based proton-sensitive field-effect transistor systems.

An NH4Cl-superfused system for a cell-cultured pH-sensing transistor was developed for detecting ion leakage across the plasma membranes of model HepG2 cells. The screening of chemical species by the method developed and conventional membrane-leakage assays identified the types of membrane injuries: structural membrane disruption and pore formation. Apoptosis-mediated membrane disordering was detected by continuously monitoring the ion-barrier breakdown of the membranes using the transistor system for an extended period. Comparisons of the ISFET assay with conventional cytotoxicity assays distinguished the cell death by direct membrane injury from that by other organelle damage. Our cell-based transistor system is fast and sensitive to ion leakage of the plasma membrane due to the small hydrodynamic size of the proton and ammonium ions as the indicators. The combination of the ion leakage assay with the existing cytotoxicity assays is a new way of classifying membrane injury and cell death induced by external chemical stimuli.

Specific Recognition of Human Influenza Virus with PEDOT Bearing Sialic Acid-Terminated Trisaccharides.

Conducting polymers are good candidates for biosensor applications when molecular recognition element is imparted. We developed trisaccharide-grafted conducting polymers for label-free detection of the human influenza A virus (H1N1) with high sensitivity and specificity. A 3,4-ethylenedioxythiophene (EDOT) derivative bearing an oxylamine moiety was electrochemically copolymerized with EDOT. The obtained film was characterized by cyclic voltammetry, X-ray photoelectron spectroscopy, scanning electron microscopy, stylus surface profilometer, and AC-impedance spectroscopy. The trisaccharides comprising Sia-α2,6'-Gal-Glu (2,6-sialyllactose) or Sia-α2,3'-Gal-Glu (2,3-sialyllactose) were covalently introduced to the side chain of the conducting polymers as a ligand for viral recognition. Immobilization of sialyllactose was confirmed by quartz crystal microbalance (QCM) and water contact angle measurements. Specific interaction of 2,6-sialyllactose with hemagglutinin in the envelope of the human influenza A virus (H1N1) was detected by QCM and potentiometry with enhanced sensitivity by 2 orders of magnitude when compared with that of commercially available kits. The developed conducting polymers possessing specific virus recognition are a good candidate material for wearable monitoring and point-of-care testing because of their processability and mass productivity in combination with printing technologies.