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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.

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.

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...

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.

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.

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.

Solid-state electrochemical sensors are developing as a new platform for liquid biopsy, combining detection and analysis of nucleic acids with isothermal nucleic acid amplification reactions. For the detection of small nucleic acids such as cfDNA, ctDNA and miRNA in liquid biopsy, further development of highly sensitive and precise devices is required to transcend the barrier of classical detection technology. Electrochemical sensors based on engineering approaches are advantageous for miniaturization of systems and convenience of diagnosis, and the analysis of data obtained with these sensors dovetails with transition to an information society. This review briefly surveys the current development of electrochemical sensors combined with isothermal amplification for nucleic acid detection and predicts the future direction of research.

In an attempt to develop a totally synthetic, self-regulated insulin delivery system to treat diabetes, N-substituted poly(acrylamide) copolymer gels bearing boronate functionality have been chemic...

A group of heterocyclic boronic acids demonstrating unusually high affinity and selectivity for sialic acids are described, with strong interactions under the weakly acidic pH conditions associated with a hypoxic tumoral microenvironment., Boronic acids are well known for their ability to reversibly interact with the diol groups found in sugars and glycoproteins. However, they are generally indiscriminate in their binding. Herein we describe the discovery of a group of heterocyclic boronic acids demonstrating unusually high affinity and selectivity for sialic acids (SAs or N-acetylneuraminic acid), which are sugar residues that are intimately linked with tumor growth and cancer progression. Remarkably, these interactions strengthen under the weakly acidic pH conditions associated with a hypoxic tumoral microenvironment. In vitro competitive binding assays uncovered a significantly higher ability of 5-boronopicolinic acid, one of the derivatives identified in this work as a strong SA-binder, to interact with cell surface SA in comparison to a gold-standard structure, 3-propionamidophenylboronic acid, which has proven to be an efficient SA-binder in numerous reports. This structure also proved to be suitable for further chemical conjugation with a well-preserved SA-binding capability. These findings suggest an attractive alternative to other ongoing boronic acid based chemistry techniques aiming to achieve tumor-specific chemotherapies and diagnoses.

Ligand-mediated targeting of nanocarriers to tumors is an attractive strategy for increasing the efficiency of chemotherapies. Sialylated glycans represent a propitious target as they are broadly overexpressed in tumor cells. Because phenylboronic acid (PBA) can selectively recognize sialic acid (SA), herein, we developed PBA-installed micellar nanocarriers incorporating the parent complex of the anticancer drug oxaliplatin, for targeting sialylated epitopes overexpressed on cancer cells. Following PBA-installation, the micelles showed high affinity for SA, as confirmed by fluorescence spectroscopy even at intratumoral pH conditions, i.e., pH 6.5, improving their cellular recognition and uptake and enhancing their in vitro cytotoxicity against B16F10 murine melanoma cells. In vivo, PBA-installed micelles effectively reduced the growth rate of both orthotopic and lung metastasis models of melanoma, suggesting the potential of PBA-installed nanocarriers for enhanced tumor targeting.

Stimuli-responsive “smart gels” that undergo physicochemical property changes in response to external stimuli can provide both a functional and structural basis for selfregulated materials and systems. Of particular interest are those materials sensitive to chemical stimuli, that is, the fluctuation in the concentration of specific biomolecules, that can mimic biofeedback systems, thus providing many attractive platforms for biomedical applications. Glucose has been a molecule of interest for decades owing to its relevance to the treatment of diabetes. Diabetes is not an infectious disease, but its rapidly increasing worldwide prevalence has been recognized as a pandemic, and thus diabetes poses a serious global health threat much like epidemics of truly infectious diseases, such as HIV/AIDS. Despite the necessity for the continuous and accurate glycemic control in the management of insulin-dependent diabetes mellitus (IDDM), the current palliative treatment relies almost solely on the patients self injection of insulin; this self injection not only impinges on the quality of life of the patient but also fails to precisely control the dose of insulin, where an overdose must be strictly avoided as it causes serious hypoglycemia. For the preparation of self-regulated insulin delivery systems, the following two approaches have historically prevailed. One is based on enzymatic reactions between glucose oxidase (GOD) and glucose, a similar rationale to those exploited in commercial glucose sensors. The other type utilizes carbohydrate-binding lectin proteins, such as concanavalin A (Con A), as a complementary binder to glucose. These protein-based materials eventually denature and lose activity, and thus are not practical for long-term use and storage. Another problem is the strong cytotoxicity of Con A; this cytotoxicity has, thus far, prevented any clinical applications of these materials. Herein we describe a totally synthetic alternative to these materials. Phenylboronic acid (PBA), a synthetic molecule capable of reversibly binding with 1,2or 1,3-cis-diols including glucose, is utilized as the molecular basis. Our previous studies have shown that the glucose-dependent shift in the equilibrium of PBA between the uncharged and anionically charged forms, when coupled with a properly amphiphilic three-dimensional backbone (or gel), could induce a reversible change in the volume of the gel. The resultant abrupt and rapid change in the hydration, under certain conditions, could cause a localized dehydration of the gel surface, that is, a so-called skin layer, thus offering a method to instantly control the permeation of gel-loaded insulin. This goal is a great challenge and relies on achieving sufficient glucose sensitivity under physiological pH and temperature, that is, pH 7.4 and 37 8C, while also fine tuning the system so it shows a gated response to the change in glucose concentration critically at the level of normoglycemia, i.e., ca. 1 gl . To achieve this goal, we have systemically explored the structure–property correlation by focusing on (meth)acrylamide-based hydrogels. The major efforts have been directed to controlling the apparent pKa of PBA; the pKa depends not only on chemical structure of PBA, but also on the state of hydration. To make the situation more complicated, in a hydrogel environment the degree of hydration is a function of (as well as temperature, ionic strength, pH, and sugar concentration) the hydrophilicity, rigidity, and density of the main chain components and the amount of PBA. Herein we describe a gel that meets all the above criteria; the chemical structure of this gel turned out to be a remarkably simple copolymer system. A smart gel has been shown to act as an artificial pancreas under conditions closely related to human glucose homeostasis. As illustrated in Figure 1a, PBA derivatives in water exist in equilibrium between uncharged (i) and anionically charged (ii) forms. Upon addition of glucose, only the charged PBA (ii) can form a stable complex with glucose (iii), and the formation of this complex results in an apparent decrease in the amount of anionically charged PBA (ii). On consumption of the phenylboronate (ii), the equilibrium between (i) and (ii) shifts toward the latter (ii). Since the complexed PBA (iii) is also anionically charged, further addition of glucose leads to a shift in the equilibrium (i + ii + iii) toward the phenylboronate anions (ii + iii), and a decrease in the amount of [*] Dr. A. Matsumoto, J. Nishida, Dr. H. Matsumoto, Dr. Y. Miyahara Institute of Biomaterials and Bioengineering Tokyo Medical and Dental University Kanda-surugadai 2-3-10, Chiyoda-ku, Tokyo 101-0062 (Japan) E-mail: miyahara.bsr@tmd.co.jp Dr. T. Ishii Department of Bioengineering, The University of Tokyo Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8656 (Japan)

Beta-nicotinamide adenine dinucleotide (NAD(+)) and its reduced form (NADH) play major roles in the development of electrochemical enzyme biosensors and biofuel cells. Unfortunately, the oxidation of NADH at carbon electrodes suffers from passivation of the electrodes and a decrease in passing currents. Here, we investigate experimentally and theoretically the reasons for such passivation. High-resolution X-ray photoelectron spectroscopy (HR-XPS), voltammetry, and amperometry show that adsorption occurs on the edges and "edge-like" defects of graphene sheets. HR-XPS and ab initio molecular dynamics show that the adsorption of NAD(+) molecules on the edges of graphene happens due to interaction with oxygen-containing groups such as carboxylic groups, while graphene edges substituted only with hydrogen are prone to passivation.

A comprehensive basicity study of alpha,omega-alkanediamines and related bases has been carried out. Basicities in acetonitrile (AN, pK(a) values), tetrahydrofuran (THF, pK(alpha) values), and gas phase (GP, GB values), were measured for 16, 14, and 9 diamine bases and for several related monoamines. In addition the gas-phase basicities and equilibrium geometries were computed for 19 diamino bases and several related monoamines at the DFT B3LYP 6-311+G** level. The effects of the different factors (intrinsic basicity of the amino groups, formation of intramolecular hydrogen bonds, and molecular strain) determining the diamine basicities were estimated by using the method of isodesmic reactions. The results are discussed in terms of molecular structure and solvation effects. The GP basicity is determined by the molecular size and polarizability, the extent of alkylation, and the energy effect of intramolecular hydrogen bond formation in the protonated base. The basicity trends in the solvents differ very much from those in GP: 1) The solvents severely compress the basicity range of the bases studied (3.5 times for the 1,3-propanediamine family in AN, and 7 times in THF), and 2) while stepwise alkylation of the basicity center leads to a steady basicity increase in the gas phase, the picture is complex in the solvents. Significant differences are also evident between THF and AN. The high hydrogen bond acceptor strength of THF leads to this solvent favoring the bases with "naked" protonation centers. In particular, the basicity order of N-methylated 1,3-propanediamines is practically inverse to that in the gas phase. The picture in AN is intermediate between that of GP and THF.

2-Methacryloyloxyethyl phosphorylcholine (MPC) is a custom methacrylate with a zwitterionic phosphorylcholine moiety on the side chain. In the past 25 years, MPC has been used as a building block for a wide range of polymeric biomaterials because of its excellent resistance to nonspecific protein adsorption, cell adhesion, and blood coagulation. Recently, MPC polymers with specific features have been used in bioengineering and nanomedicine. This review focuses on three topics that highlight the latest findings on MPC polymers, that is, specific recognition of C-reactive protein (CRP), cell-membrane-penetration abilities, and lubrication properties. These developments will extend the applications of this biomimetic material from bioinert polymers to biosensing, CRP inhibitors, prodrug carriers, subcellular bioimaging, cell manipulation, and joint replacement. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015, 132, 41766.

We developed a quantitative detection scheme for nucleic acids, combining solid-phase rolling circle amplification and chronocoulometry (RCA-CC). A gold electrode was directly formed on a polystyrene substrate as a cost-effective and flexible biosensor for sensitive detection of microRNA (mir-143) in blood samples.

The next higher homologue of hexamethylenetetramine was synthesized as the proton cryptate H+ @1⋅Br- (shown schematically), and its X-ray structure determined. The proton trapped by the lone pairs accumulated at the center of the T-symmetric tetraaza cage could not be exchanged or removed, even after heating for three days in 3 M NaOD.

Here described is a phenylboronic acid (PBA) based glucose-responsive hydrogel operating under physiological pH and temperature, a material potentially applicable to a totally synthetic smart insulin delivery system to treat diabetes.

The replica molding and embossing of DNA texture has been achieved using a conventional polyurethane mold. The final process requires no additional or separate extraction phases. The polyurethane replica is stable up to 150 degrees C and possesses a good longevity and a capacity to emboss a biological entity into a thermosetting plastic such as poly(ethylene terephthalate).

Alternations of sialic acid (SA) content on cell surface glycan chains have been implicated in numerous normal and pathological processes including developments, differentiations, and tumor metastasis. Overexpressions of SA have been implicated in the malignant and metastatic phenotypes for many different types of cancers, while decreased SA expression has also been identified in erythrocytes of diabetic mellitus. Techniques to conveniently monitor cell surface SA would therefore have great relevance to cytology. Preexisting methodologies to quantify SA, however, involve multiple enzymatic, dye-labeling, and lethal procedures, which are costly and time-consuming. Here we developed a potentiometric SA detection using a phenylboronic acid (PBA) compound integrated into the form of a self-assembled monolayer (SAM) onto a field effect transistor (FET) extended gold gate electrode. Due to selective binding between undisassociated PBA and SA at pH 7.4 among other glycan chain constituent monosaccharides, we found that carboxyl anions of SA were exclusively detectable as the change in threshold voltage (V(T)) of the PBA-modified FET. The technique was applied to analyses of altered SA expressions on rabbit erythrocyte as a model for diabetes. Comparative SA expression analyses for each healthy and diseased model revealed that the disease could be feasibly diagnosed simply by placing the known-count cell suspensions onto the device without any labeling and enzymatic procedures. Such a technique may also provide a quantitative adjunct to histological evaluation of tumor malignancy and metastatic potential during intra- and postoperative diagnoses. Also advantageously, a technique herein described is all within a CMOS (Complementary Metal Oxide Semiconductor) compatible format thus promising for highly efficient and low cost manufacturing with readiness of downsizing and integration by virtue of advanced semiconductor processing technologies.

We developed a new building block for a protein- and cell-repellant self-assembled monolayer (SAM) from 2-methacryloyloxyethyl phosphorylcholine (MPC) via a simple Michael-type addition to one mercapto group in alkanedithiol. The thiolated MPC can enable functionalization of a noble metal electrode to minimize noise signal in biosensing.

We report a method for detecting microRNAs encapsulated in exosomes using a microelectrode array in semiconductor-based potentiometry after RT-PCR. The inherent miniaturization of the electrical biosensor meets requirements for massively parallel analysis of circulating microRNA as a noninvasive biomarker.

A novel cyclam-like macrocyclic tetramine 10 with two bispidine units was synthesized by coupling of bispidine 7 with bispidine bis(α-chloroacetamide) 8, followed by reduction with DIBAL-H. The tetramine 10 revealed very strong basicity and lithium coordinating ability.

An ultrathin coating of individual multi-wall carbon nanotubes with germanium oxide was developed. Coated carbon nanotubes were prepared by in situ gelation of tetramethoxy germanium, forming an amorphous germanium oxide layer. The resulting germanium oxide/carbon nanotube nanocables were characterized by transmission electron microscopy (TEM), TEM/energy-dispersive x-ray spectroscopy, x-ray photoelectron spectroscopy (XPS) and current-voltage measurements. The approximately 10 nm thin germanium oxide layer provides excellent electrical insulating properties for individual carbon nanotubes.

A field effect transistor (FET) whose gate surface has been modified with a stimuli-responsive "smart" polymer gel can visualize the kinetics of the chemo-mechanical signal transduction as a mode of its altered electrical characteristics without any labels.

The reaction of benzylamine with 2,6-bis(bromomethyl)pyridine has now been found to provide the trimeric title compound as the major product instead of the reported dimer. When the reaction was conducted in two steps, a new tetrameric pyridinophane was isolated as the major product.

Effects of fluorine substituents on transannular interaction in [3.3]metacyclophanes were examined on the basis of chemical and spectral properties of five [3.3]metacyclophanes possessing one or two fluorine atoms at the inner and/or outer positions.

A new kind of solid-state contact for ion-selective electrodes based on solvent polymeric membranes is described. These electrodes show similar electrochemical behaviour to conventional ion-selective membranes in contact with an internal filling solution. They exhibit no significant interference with oxygen, nitrogen or carbon dioxide.

Initially described are several improvements in the synthesis of dodecahedrane precursors and dodecahedrane (XIII) itself.

The isothermal amplification of DNA in minimally buffered conditions allows to perform and monitor nucleic acid amplification with minimal technological and operative requirements. We show in this work how phi29 can operate multiple displacement amplification in minimally buffered conditions producing, as a readout, pH shifts attaining subunits of pH.

The base-catalyzed reaction of diketosulfides (RCOCH2)2S (R=phenyl, p-tolyl) with biacetyl or benzil have provided high yields of 2,5-diacyltetrahydrothiophene-3,4-diol derivatives rather than the expected 2,5-diacylthiophenes. The diols have been transformed cleanly into the corresponding 2,5-diacylthiophenes by treatment with thionyl chloride-pyridine. In contrast, the reaction of the diketo sulfide (R=p-tolyl) with phenylglyoxal afforded a 2,5-diacylthiophene directly in good yield.

The Wurtz reaction has been found to be catalyzed by easily removable tetrakis(p-dimethylaminophenyl)ethylene. This modification facilitated the separation of products in the syntheses of [2.1.2.1.]-, [2.2.2.2]-, and [3.2.3.2]paracyclophanes, the isolated yields of which were better than in the case of tetraphenylethylene.