Influence of patterned concave depth and surface curvature on anodization of titania nanotubes and alumina nanopores

Vertically aligned TiO(2) nanotube and Al(2)O(3) nanopore arrays have been obtained by pattern guided anodization with uniform concave depths. There are some studies about the effect of surface curvature on the growth of Al(2)O(3) nanopores. However, the surface curvature influence on the development of TiO(2) nanotubes is seldom studied. Moreover, there is no research about the effect of heterogeneous concave depths of the guiding patterns on the anodized TiO(2) nanotube and Al(2)O(3) nanopore characteristics, such as diameter, growth direction, and termination/bifurcation. In this study, focused ion beam lithography is used to create concave patterns with heterogeneous depths on flat surfaces and with uniform depths on curved surfaces. For the former, bending and bifurcation of nanotubes/nanopores are observed after the anodization. For the latter, bifurcation of a large tube into two smaller tubes occurs on concave surfaces, while termination of existing tubes occurs on convex surfaces. The growth direction of all TiO(2) nanotubes is perpendicular to the local surface and thus is different on different facets of the same Ti foil. At the edge of the Ti foil where two facets meet, the nanotube growth direction is bent, resulting in a large stress release that causes the formation of cracks.     

PCR-free electrochemical assay of telomerase activity

In this paper, we report a non-PCR-based electrochemical assay that can detect telomerase activity. Telomerase from HeLa cells may induce telomerization of thiolated primers immobilized on a gold electrode surface. With the telomerization reaction, more and more guanine-rich telomeric repeats are formed, so the electrochemical oxidation signal of guanine at about 1.00 V, which is utilized to indicate the elongated guanine-rich telomeric repeats tethered to the primers, will be increased. This assay method can detect the telomerase activity originated from 3000 HeLa cells and thus holds promise as a simple and sensitive approach in clinical diagnosis of cancer.     

Carbohydrate monolayer strategy for electrochemical assay of cell surface carbohydrate

The study of glycobiology has been seriously hampered due to lack of an ideal assay tool. This work proposes a robust carbohydrate monolayer platform to solve the problems of active site inaccessibility and lectin denaturation associated with protein arrays reported for detection of cell surface carbohydrates and develops a convenient method for monitoring cell surface carbohydrate sites of interest, with high sensitivity, acceptable rapidity, low cost, and excellent extensibility. It utilizes the competitive binding of solid-surface-confined and cell-surface-residing carbohydrates to quantum dot labeled carbohydrate recognition protein and subsequent voltammetric quantification of the metal signature. The mannan monolayer strategy exhibited sensitive response to K562 cells and possessed potential specificity due to the specific interaction between lectin and corresponding carbohydrate. By comparing the competitive binding of K562 cells with mannan in solutions, the average Con A binding capacity of a single K562 cell could be estimated to correspond to 6.9 pg or 2.3 x 10(10) mannose moieties. This strategy integrates the advantages of surface assembly, nanotechnology, bioconjugate techniques, and electrochemical detection and can be expanded for profiling cell surface carbohydrates and high-throughput multiple detection by simultaneously using more pairs of lectin and carbohydrate owing to the multiple coding capability of QDs, which provides an important protocol for the quantitative evaluation of cell surface carbohydrate sites.     

Synthesis of a head-to-tail-type cyclodextrin-based insulated molecular wire

We developed a new method for synthesizing an organic-soluble insulated molecular wire (IMW) using permethylated cyclodextrin (PMCD). The IMW obtained using this method is highly soluble in a variety of organic solvents and has a high covering ratio, regioregularity, rigidity, photoluminescence efficiency, and interchain hole mobility.     

Protein nanopores with covalently attached molecular adapters

Molecular adapters are crucial for the stochastic sensing of organic analytes with alpha-hemolysin (alphaHL) protein nanopores when direct interactions between analytes and the pore cannot readily be arranged by conventional protein engineering. In our earlier studies, cyclodextrin adapters were lodged noncovalently within the lumen of the alphaHL pore. In the present work, we have realized the controlled covalent attachment of a beta-cyclodextrin (betaCD) adapter in the two possible molecular orientations inside alphaHL pores prepared by genetic engineering. There are two advantages to such a covalent system. First, the adapter cannot dissociate, which means there are no gaps during stochastic detection, a crucial advance for single-molecule exonuclease DNA sequencing where the continuous presence of a molecular adapter will be essential for reading individual nucleotides. Second, the ability to orient the adapter allows analytes to bind through only one of the two entrances to the betaCD cavity. We demonstrate that the covalently attached adapters can be used to alter the ion selectivity of the alphaHL pore, examine binding events at elevated temperatures, and detect analytes with prolonged dwell times.     

Highly sensitive assay for gamma-glutamyltranspeptidase activity by high-performance liquid chromatography with electrochemical detection

A highly sensitive assay for gamma-glutamyltranspeptidase activity involving high-performance liquid chromatography (HPLC) with electrochemical detection was devised. gamma-Glutamyl-DOPA, a new synthetic dipeptide, which consists of naturally occurring amino acids, was found to be a good substrate for gamma-glutamyltranspeptidase purified from Proteus mirabilis. Enzymatically formed DOPA was adsorbed on an aluminium oxide column, eluted with 0.5 M hydrochloric acid and determined by HPLC with electrochemical detection. The sensitivity limit of this method was 0.5 pmol of DOPA formed. Some properties of gamma-glutamyltranspeptidase purified from P. mirabilis were investigated using gamma-glutamyl-DOPA as a substrate. In the presence of 0.15 M glycylglycine, the KM value of the enzyme for gamma-glutamyl-DOPA was 0.013 mM, and the maximum velocity was 247 nmol/min per mg protein. This method was applied to the assay of the enzymatic activity in human serum.     

A boronate-modified renewable nanointerface for ultrasensitive electrochemical assay of cellulase activity

The saccharification of cellulosic biomass to produce biofuels and chemicals is one of the most promising industries for green-power production and sustainable development. Cellulase is the core component in the saccharification process. Simple and efficient assay method to determine cellulase activity in saccharification is thus highly required. In this work, a boronate-affinity surface based renewable and ultrasensitive electrochemical sensor for cellulase activity determination has been fabricated. Through boronate-sugar interaction, celluloses are attached to the electrode surface, forming the cellulose nano-network at the sensing interface. Cellulase degradation can lead to the variation of electrochemical impedance. Thus, electrochemical impedance signal can reflect the cellulase activity. Importantly, via fully utilizing the boronate-affinity chemistry that enables reversible fabrication of cellulose nano-network, a renewable sensing surface has been firstly constructed for cellulase activity assay. Thanks to interfacial diffusion process of electrochemical sensor, the product inhibitory effect in the cellulase activity assays can be circumvented. The proposed electrochemical sensor is ultrasensitive for label-free cellulase activity detection with a very simple fabrication process, showing great potential for activity screen of new enzymes in saccharification conversion.     

Highly sensitive assay for choline acetyltransferase activity by high-performance liquid chromatography with electrochemical detection

A highly sensitive assay for choline acetyltransferase activity by high-performance liquid chromatography with electrochemical detection was devised. This assay method is based on the separation of acetylcholine and choline on a Develosil Ph-5 reversed-phase column (a phenyl column), followed by their enzymatic conversion to hydrogen peroxide through post-column reaction with acetylcholinesterase and choline oxidase. The sensitivity of the system is excellent and 5 pmol of acetylcholine enzymatically formed could be detected. The linearity between the peak height and the amount of acetylcholine was observed over the range of 5 pmol to 5 nmol. Some enzymatic properties were investigated by using a soluble fraction of bovine caudate nucleus as enzyme. The Michaelis constants of the enzyme for choline and acetyl coenzyme A were 0.3 mM and 0.03 mM, respectively. The enzyme exhibited the maximum activity over the pH range 7.4-9.5. The regional distribution of choline acetyltransferase activity in rat brain was examined. The order of the activity from the highest to the lowest agreed with the reported brain distribution of the enzyme: striatum, pons plus medulla oblongata, cerebral cortex, thalamus plus hypothalamus, olfactory bulb and cerebellum.     

Simultaneous formation behaviour of surface structures and molecular alignment by patterned photopolymerisation

ABSTRACT

Highly functional soft materials with fine control of molecular alignment are of great interest for the applications in various fields. However, the current method of molecular alignment still has some challenges. Recently, we have developed a new alignment process based on a concept of scanning wave photopolymerisation (SWaP). When a sample is irradiated with spatially selected light, a polymerisation occurs only in an irradiated region, leading to a molecular motion between the irradiated and unirradiated regions. Such molecular motion generates the alignment of liquid crystal molecules. Moreover, a surface relief structure is formed in the polymer film by the molecular motion. In this study, we simultaneously formed the surface structure and molecular alignment by the patterned photopolymerisation. We compared the degree of molecular alignment with the shape of the created surface structure, and revealed that the degree of molecular alignment was maximized at the boundary of irradiated and unirradiated regions. This method enables one to form both molecular alignment patterning and surface structuring in a single step.     

Ag-polymer composite microspheres with patterned surface structures

Novel silver-poly(acrylamide-co-methacrylic acid) [Ag-P(AM-co-MAA)] composite microspheres, in tens of micrometer size range, with patterned surface and core/shell structures were prepared by chemical reduction of Ag₂CrO₄-P(AM-co-MAA) composite microspheres in ethanol. Characterization with various techniques revealed that the chemical composition of the “shell” is dominated by Ag, but the “core” is dominated by the template, P(AM-co-MAA). It was also demonstrated that the surface morphology of the Ag-polymer composite microspheres is similar to that of their precursors and can be controlled to a certain extent by varying the composition of template copolymer, approaches, and amount of Ag₂CrO₄ deposited. This morphology transfer technique is also applicable for other silver salts-polymer composite microspheres. The same silver-polymer composite microspheres with very different morphology have also been prepared by utilizing this technique, but the different precursor microspheres, Ag₃PO₄-P(AM-co-MAA), were used. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Gold polycarbonate microchannels for electrochemical applications

In this paper, we present gold-plating polycarbonate (PC) microchannels. The fabrication of the gold microfluidic channels is achieved by tuning the sequence of reagent insertion into milled and closed submillimeter PC system channels. The resulting gold surface can be utilized in many applications where the benefits of microfluidics, (bio)chemistry of surfaces, and electrochemistry can be combined. Here, we combine the advantages of electrochemistry with microfluidics by mixing the gold sensor with microfluidics. This approach differs from the classic one – the sensor will undergo modifications (e. g., shape and size) depending on the specific scientific problem and will be designed individually; hence its characteristics will be changed. Our goal in this work is to indicate new possibilities for combining two methodologies – electrochemistry and microfluidics. In our work, we emphasize that it confirms the validity of our chosen concept (proof-of-concept). In this work, we present one such application, the use of a gold microfluidic channel as a working electrode (WE). We describe the microchip‘s construction and electrochemical characterization, including the gold flow-through WE, the Ag/AgCl wire pseudo-reference, and the Pt auxiliary electrode. The measured current is the result of the flow through a rectangular duct of the gold microchannel electrode embedded in the four walls of the chip.     

Effects of surface curvature and surface chemistry on the structure and activity of proteins adsorbed in nanopores

The interactions of proteins with the surface of cylindrical nanopores are systematically investigated to elucidate how surface curvature and surface chemistry affect the conformation and activity of confined proteins in an aqueous, buffered environment. Two globular proteins, lysozyme and myoglobin, with different catalytic functions, were used as model proteins to analyze structural changes in proteins after adsorption on ordered mesoporous silica SBA-15 and propyl-functionalized SBA-15 (C(3)SBA-15) with carefully controlled pore size. Liquid phase ATR-FTIR spectroscopy was used to study the amide I and II bands of the adsorbed proteins. The amide I bands showed that the secondary structures of free and adsorbed protein molecules differ, and that the secondary structure of the adsorbed protein is influenced by the local geometry as well as by the surface chemistry of the nanopores. The conformation of the adsorbed proteins inside the nanopores of SBA-15 and C(3)SBA-15 is strongly correlated with the local geometry and the surface properties of the nanoporous materials, which results in different catalytic activities. Adsorption by electrostatic interaction of proteins in nanopores of an optimal size provides a favorably confining and protecting environment, which may lead to considerably enhanced structural stability and catalytic activity.     

Gold nanoparticle-based surface enhanced Raman scattering spectroscopic assay for the detection of protein-protein interactions

In the present work, a sensitive spectroscopic assay based on surface-enhanced Raman spectroscopy (SERS) using gold nanoparticles as substrates was developed for the rapid detection protein-protein interactions. Detection is achieved by specific binding biotin-modification antibodies with protein-stabilized 30 nm gold nanoparticles, followed by the attachment of avidin-modification Raman-active dyes. As a proof-of-principle experiment, a well-known biomolecular recognition system, IgG with protein A, was chosen to establish this new spectroscopic assay. Highly selective recognition of IgG down to 1 ng/ml in solution has been demonstrated.     

Small volume bead assay for ovalbumin with electrochemical detection

A bead based sandwich enzyme immunoassay coupled to electrochemical detection for ovalbumin has been developed. The enzyme label alkaline phosphatase was used to convert the substrate 4-aminophenyl phosphate to electroactive product 4-aminophenol. The detection was done in a microdrop by continuously monitoring the enzyme turnover with a rotating disk electrode. This reduces dilution of the enzyme product, a key to achieving low detection limits. The assay developed has a detection limit of 0.1 ng ml-1. Assay sensitivity in complex matrices such as food and serum was compared.     

A cyclodextrin-based molecular reactor to template the formation of indigoid dyes

N,N′-Bis(6^A-deoxy-β-cyclodextrin-6^A-yl)urea behaves as a molecular reactor to bias competing reactions of indoxyl anion and isatin-5-sulfonate in water, to give indigo and indirubin-5′-sulfonate. It appears that the cyclodextrin dimer increases the relative reactivity of the isatin-5-sulfonate, by selectively complexing the reactive form. The molecular host also aligns the isatinsulfonate with indoxyl anion to favour production of indirubin-5′-sulfonate, with the result that the ratio of indigo and indirubin-5′-sulfonate produced is altered by a factor of at least 3500, without a substantial loss of yield.     

An electrochemical approach for detection of DNA methylation and assay of the methyltransferase activity

This work develops an electrochemical approach for rapid detection of the genomic DNA methylation level, assay of methyltransferase activity, and evaluation and screening of the inhibitors of methyltransferase. This method may be a help for the discovery of anticancer drugs.     

The effect of temperature on the analysis of metanephrine for catechol-O-methyltransferase activity assay by HPLC with electrochemical detection

The enzyme catechol-O-methyltransferase (COMT) plays an important role in the metabolism of catechol estrogens and degradation of the catecholamine neurotransmitters, such as epinephrine. Several analytical methods, mainly high-performance liquid chromatography with electrochemical amperometric detection, have been reported for the analysis of catecholamines and their metabolites in biological fluids. In this paper we report the relevance of controlling temperature in calibration procedures of metanephrine, an O-methylated product of catechol-O-methyltransferase, using epinephrine as substrate. The results at higher temperatures show shorter retention times of metanephrine, no undue band-broadening and increased electro signals. This study also showed that, despite different temperatures leading to similarly specific activities of recombinant human COMT as expected, there are additional advantages in flow analytical methods where good sensitivity, efficiency and selectivity is required, mainly in tissues with low levels of COMT activity.     

Substrate assisted electrochemical deposition of patterned cobalt thin films

The patterned Co layers deposited on the scratched Cu surfaces were investigated with the use of the scanning electron microscopy. Patterned cobalt thin films were electrochemically deposited from the cobalt sulfate bath at room temperature. Pattering of cobalt was carried out by simple means of substrate scratching. Gentle scratching induces a direct pattering of cobalt from vertical to horizontal. The prepared pattered films were characterized for their structural, surface morphological and compositional properties by means of X-ray diffraction, scanning electron microscopy and energy dispersive spectroscopy. X-ray diffraction studies reveal that the films are of cobalt. From the SEM images fabrication of patterns of cobalt is apparent. This work demonstrates a novel approach for obtaining patterned cobalt for many technological applications.     

Preparation of metal sulfide-polymer composite microspheres with patterned surface structures

CuS-poly(N-isopropylacrylamide), CuS-poly(N-isopropylacrylamide-co-methacrylic acid), Ag(2)S-poly(N-isopropylacrylamide) and Ag(2)S-poly(N-isopropylacrylamide-co-methacrylic acid) composite microspheres exhibiting complex surface morphologies were prepared by employing the minigel template method.     

Nanoparticle displacement assay with electrochemical nanopore-based sensors

The proof of concept of a nanoparticle displacement assay that enables the use of large diameter nanopores for the detection of targets of smaller molecular dimensions is presented. We hypothesized that an inherent signal amplification should arise from the selective displacement of nanoparticles preloaded in a nanopore by a much smaller molecular target. The method is demonstrated using peptide nucleic acid (PNA)-functionalized gold nanopore arrays in which short DNA-modified gold nanoparticles are anchored by weak interaction. Complementary microRNAs are detected via the resistance change caused by competitive displacement of nanoparticles from the PNA-functionalized nanopores.