Construction of amperometric immunosensors based on the electrogeneration of a permeable biotinylated polypyrrole film

The construction of amperometric immunosensors to cholera antitoxin immunoglobulins were shown to have improved sensitivity when the cholera toxin B subunit biorecognition entity was linked to an electrogenerated biotinylated polypyrrole film copolymerized with pyrrole-lactobionamide monomer. The copolymer exhibits greater film permeability than biotinylated polypyrrolic or polyphenolic films for the permeation of electroactive species. Hence, when the presence of the HRP marker of the immunoassay was determined using hydroquinone, the production of electroactive quinone was shown to permeate faster to the electrode, thus providing a faster response time.     

Microfluidic one-step synthesis of alginate microspheres immobilized with antibodies

Micrometre- and submicrometre-size functionalized beads are frequently used to capture targets of interest from a biological sample for biological characterizations and disease diagnosis. The main challenge of the microbead-based assay is in the immobilization of probe molecules onto the microbead surfaces. In this paper, we report a versatile droplet microfluidics method to fabricate alginate microspheres while simultaneously immobilizing anti-Mycobacterium tuberculosis complex IgY and anti-Escherichia coli IgG antibodies primarily on the porous alginate carriers for specific binding and binding affinity tests. The binding affinity of antibodies is directly measured by fluorescence intensity of stained target bacteria on the microspheres. We demonstrate that the functionalized alginate microspheres yield specificity comparable with an enzyme-linked immunosorbent assay. The high surface area-to-volume ratio of the functionalized porous alginate microspheres improves the detection limit. By using the droplet microfluidics, we can easily modify the size and shape of alginate microspheres, and increase the concentration of functionalized alginate microspheres to further enhance binding kinetics and enable multiplexing.     

Athermal arrayed waveguide gratings in silicon-on-insulator by overlaying a polymer cladding on narrowed arrayed waveguides

Athermal arrayed waveguide gratings (AWGs) in silicon-on-insulator (SOI) are experimentally demonstrated for the first time to our knowledge. By using narrowed arrayed waveguides, and then overlaying a polymer layer, the wavelength temperature dependence of the AWGs is successfully reduced to -1.5 pm/°C, which is more than 1 order of magnitude less than that of normal SOI AWGs. The athermal behavior of the AWGs is obtained with little degradation of their performance. For the central channel, the cross talk is less than -15 dB and the insertion loss is around 2.6 dB. Good characteristics can be maintained with temperatures up to 75 °C. The total size of the device is 350 μm × 250 μm.     

Electrochemical behavior of ascorbate oxidase immobilized on graphite electrode modified with Au-nanoparticles

Direct electrochemistry of ascorbate oxidase was observed when immobilized on graphite modified with nano-sized gold structures. Au-structures were electrodeposited onto the graphite surface by means of cyclic voltammetry, then the enzyme was chemisorbed onto their surface. The electron transfer between the enzyme active center and the modified electrode surface was probed by square wave voltammetry (SWV) and cyclic voltammetry (CV). The dependence of the current maxima on the scan rate was found linear, suggesting that the redox process is controlled by surface chemistry. Bioelectrocatalytic oxidation of the enzyme substrate l-ascorbic acid was explored by constant potential amperometry over the potential range from 200 to 350 mV (vs. Ag/AgCl, 3 M KCl) at the рНs 5.6 and 7.0. At a potential as low as 200 mV, pH 7.0 and temperature 25 °C following operational parameters were determined for the enzyme electrode: a sensitivity: 1.54 μA mM⁻¹ mm⁻² (r² = 0.99₅), linear dynamic range up to 3.3 mМ, detection limit of 1.5 μМ, response time up to 20 s.     

Fabrication and testing of trilayers with a high deposition rate plasma-polymerized spacer

A great deal of effort has recently been directed toward the development of digital optical storage systems using Ga-Al-As lasers for both reading and writing. The relatively low output power of these devices necessitates sensitive optical storage media if reasonable data rates are to be achieved. At the same time, it is desirable that the material exhibit archival properties. One method of reducing the writing energy for a wide variety of materials is to incorporate them within an optical structure that improves the optical efficiency. In particular, trilayer structures consisting of an aluminum layer 30 nm thick, a spacer layer about 100 nm thick and a thin storage layer offer the potential of improved sensitivity for a wide range of active materials, but their use requires the fabrication of a low thermal conductivity optically clear spacer layer approximately one-quarter wave thick. Because of manufacturing considerations, it is desirable that this layer be deposited at relatively high rates in an inexpensive process. Here we describe the investigation of the plasma polymerization of various monomers for this purpose. It has been found that stable films of perfluoro-2-butene can be deposited at rates greater than 1 nm s^-1. Trilayer structures using this material as the spacer layer are compared with more conventional materials.     

Electrochemical behavior of polyphenol oxidase immobilized in self-assembled structures layer by layer with cationic polyallylamine

We report here a novel bioelectrode based on self-assembled multilayers of polyphenol oxidase intercalated with cationic polyallylamine built up on a thiol-modified gold surface. We use an immobilization strategy previously described by Hodak J. et al. (Langmuir 1997, 13, 2708-2716) Quartz crystal microbalance with electroacustic impedance experiments were carried out to follow quantitatively the multilayer film formation. The response of the self-assembly polyphenol oxidase-polyallylamine electrodes toward different metabolically related catecholamines was studied, to evaluate enzyme kinetics. For the analyzed compounds, only dopamine and its metabolite Dopac gave catalytic currents at applied potential close to 0 V. These responses were proportional to the number of polyphenol oxidase-immobilized layers and were also controlled by the enzymatic reaction. The combination of microgravimetric and electrochemical techniques allowed us to determine the kinetic enzymatic constants, showing that the decomposition rate for the enzyme-substrate complex is slower than the enzymatic reoxidation step.     

Electrochemical properties of self-assembled cytochrome c on gold substrate patterned with a photosensitive polyimide film

Photosensitive polyimide having benzene and sulfonyloxyimide moieties was prepared and microarray pattern on gold substrate was obtained by a UV lithographic technique. The well-characterized monolayer of cytochrome c was immobilized on the patterned gold substrate by self-assembly process. The bioelectrochemical activity between cytochrome c molecular center and electrode interface for the self-assembled cytochrome c monolayer was investigated through the measurement of cyclic voltammetry. The integration and morphology of cytochrome c molecule through the measurement of AFM was also examined.     

Quantitative chemical derivatization technique in time-of-flight secondary ion mass spectrometry for surface amine groups on plasma-polymerized ethylenediamine film

A chemical derivatization technique in time-of-flight secondary ion mass spectrometry (TOF-SIMS) has been developed to quantify the surface density of amine groups of plasma-polymerized ethylenediamine thin film deposited on a glass surface by inductively coupled plasma chemical vapor deposition. Chemical tags of 4-nitrobenzaldehyde or pentafluorobenzaldehyde were hybridized with the surface amine groups and were detected in TOF-SIMS spectra as characteristic molecular secondary ions. The surface amine density was controlled in a reproducible manner as a function of deposition plasma power and was also quantified using UV-visible spectroscopy. A good linear correlation was observed between the results of TOF-SIMS and UV-visible measurements as a function of plasma power. This shows that the chemical derivatization technique in TOF-SIMS analysis would be useful in quantifying the surface density of specific functional groups that exist on the organic surface.     

Microfluidic enzyme immunoassay using silicon microchip with immobilized antibodies and chemiluminescence detection

Silicon microchips with immobilized antibodies were used to develop microfluidic enzyme immunoassays using chemiluminescence detection and horseradish peroxidase (HRP) as the enzyme label. Polyclonal anti-atrazine antibodies were coupled to the silicon microchip surface with an overall dimension of 13.1 x 3.2 mm, comprising 42 porous flow channels of 235-microm depth and 25-microm width. Different immobilization protocols based on covalent or noncovalent modification of the silica surface with 3-aminopropyltriethoxysilane (APTES) or 3-glycidoxypropyltrimethoxysilane (GOPS), linear polyethylenimine (LPEI, MW 750,000), or branched polyethylenimine (BPEI, MW 25,000), followed by adsorption or covalent attachment of the antibody, were evaluated to reach the best reusability, stability, and sensitivity of the microfluidic enzyme immunoassay (microFEIA). Adsorption of antibodies on a LPEI-modified silica surface and covalent attachment to physically adsorbed BPEI lead to unstable antibody coatings. Covalent coupling of antibodies via glutaraldehyde (GA) to three different functionalized silica surfaces (APTES-GA, LPEI-GA, and GOPS-BPEI-GA) resulted in antibody coatings that could be completely regenerated using 0.4 M glycine/HCl, pH 2.2. The buffer composition was shown to have a dramatic effect on the assay stability, where the commonly used phosphate buffer saline was proved to be the least suitable choice. The best long-term stability was obtained for the LPEI-GA surface with no loss of antibody activity during one month. The detection limits in the microFEIA for the three different immuno surfaces were 45, 3.8, and 0.80 ng/L (209, 17.7, and 3.7 pM) for APTES-GA, LPEI-GA, and GOPS-BPEI-GA, respectively.     

Electrochemical behaviour of molybdenum coated with flame CVD polycrystalline diamond film

Although natural diamond is a complete chemically-inert material for a wide range of aggressive environments, its comparative scarcity and problems for coating design have hampered its utility as a corrosion protective coating. The recent discovery and development of chemical vapour deposition methods for growing diamond crystals and polycrystalline diamond films has opened up a wide range of applications thanks to their excellent tribological, electronic and optical properties. Various applications are in progress for corrosion and combined wear and corrosion protection. This paper presents the first study of the corrosion behaviour of continuous polycrystalline diamond films using electrochemical impedance electroscopy. Diamond films have been deposited on molybdenum substrates by means of the acetylene flame combustion method (FCVD). Electrochemical behaviour has been studied in a 0.6 M NaCl solution, it being seen that despite the inert character and apparent continuity of the film, there are areas of the base material which are exposed to the electrolyte. This behaviour has been modelled by means of an equivalent circuit which allows for the corroboration of the proposed mechanism.     

Electrochemical preparation of Fe-Mn alloy film in organic bath

Fe-Mn alloy films have been prepared by electrodeposition in an organic bath containing FeCl₂ + MnCl₂ in dimethyl formamide. The electroreduction of Mn(II) was irreversible and the diffusion coefficient of Mn(II) was calculated to be 8.0 × 10^− 11 m² s^− 1 at 298 K. An amorphous film of Fe-Mn was obtained by potentiostatic electrolysis. The Mn content varied from 4.8 at.% to 72.3 at.% with increase in the applied cathodic potential. Scanning electron microscope investigation showed that the deposited film was homogeneous and consisted of spherical particles. Nano-sized pores were observed in the surface of these particles. After heat treatment at 773 K, large crystal grains formed and X-ray diffraction patterns indicate that solid solution of Mn in γ-Fe occurred. The alloying temperature of the Fe-Mn film was determined to be 1013 K using differential thermal analysis.     

Electrochemical protection of thin film electrodes in solid state nanopores

Solid state nanopores are a core element of next-generation single molecule tools in the field of nano-biotechnology. Thin film electrodes integrated into a pore can interact with charges and fields within the pore. In order to keep the nanopore open and thus functional electrochemically induced surface alteration of electrode surfaces and bubble formation inside the pore have to be eliminated. This paper provides electrochemical analyses of nanopores drilled into TiN membranes which in turn were employed as thin film electrodes. We studied physical pore integrity and the occurrence of water decomposition yielding bubble formation inside pores by applying voltages between -4.5 and +4.5 V to membranes in various protection stages continuously for up to 24 h. During potential application pores were exposed to selected electrolyte-solvent systems. We have investigated and successfully eliminated electrochemical pore oxidation and reduction as well as water decomposition inside nanopores of various diameters ranging from 3.5 to 25 nm in 50 nm thick TiN membranes by passivating the nanopores with a plasma-oxidized layer and using a 90% solution of glycerol in water as KCl solvent. Nanopore ionic conductances were measured before and after voltage application in order to test for changes in pore diameter due to electrochemical oxidation or reduction. TEM imaging was used to confirm these observations. While non-passivated pores were electrochemically oxidized, neither electrochemical oxidation nor reduction was observed for passivated pores. Bubble formation through water decomposition could be detected in non-passivated pores in KCl/water solutions but was not observed in 90% glycerol solutions. The use of a protective self-assembled monolayer of hexadecylphosphonic acid (HDPA) was also investigated.     

Discriminative detection of volatile sulfur compound mixtures with a plasma-polymerized film-based sensor array installed in a humidity-control system

We demonstrated the discrimination of volatile sulfur compound mixtures with different mixing ratios by using an array of the plasma-polymerized film (PPF)-coated quartz crystal resonators. The PPF sensor array, which contains PPFs prepared from amino acids and synthetic polymers, exhibited different response patterns to mono or mixed volatile sulfur compounds (VSCs) (hydrogen sulfide and methanethiol) under a dry environment. The sensor array was installed in a desktop-size relative humidity controller. The relative humidity and temperature conditions of the sample flow to the sensor cell were equalized to those of the inner atmosphere of the sensor cell based on the concept of the two-separate-temperatures method. In this way, the baseline drift of PPF sensor response caused by the introduction of a highly humid sample was successfully suppressed. We compared the sensor array responses under the controlled humidity conditions. Presorption of water molecules by PPFs caused a decrease of sensor sensitivity, but the films still had the ability to discriminate sub-ppmv VSC mixtures having 6:1, 1:1, and 1:6 mixture ratios of hydrogen sulfide and methanethiol.     

Nucleotide identification and orientation discrimination of DNA homopolymers immobilized in a protein nanopore

Nanopores have been used as extremely sensitive resistive pulse sensors to detect analytes at the molecular level. There has been interest in using such a scheme to rapidly and inexpensively sequence single molecules of DNA. To establish reference current levels for adenine, cytosine, and thymine nucleotides, we measured the blockage currents following immobilization of single-stranded DNA polyadenine, polycytosine, and polythymine within a protein nanopore in chemical orientations in which either the 3' or the 5' end enters the pore. Immobilization resulted in low-noise measurements, yielding sharply defined current distributions for each base that enabled clear discrimination of the nucleotides in both orientations. In addition, we find that not only is the blockage current for each polyhomonucleotide orientation dependent, but also the changes in orientation affect the blockage currents for each base differently. This dependence can affect the ability to resolve polyadenine and polythymine; with the 5' end entering the pore, the separation between polyadenine and polythymine is double that observed for the 3' orientation. This suggests that, for better resolution, DNA should be threaded through the 5' end first in nanopore DNA sequencing experiments.     

Electrochemical impedance study of TiAlN film coating on a Ni-based alloy in 0.9% NaCl

TiAlN films were deposited on Ni-based alloy by RF sputtering technique. Electrochemical impedance spectroscopy was employed as in situ technique to investigate the evolution of the TiAlN films on exposure in biological media. After immediate- to short-term exposure, the biological media was further evaluated with inductively coupled plasma-atomic emission spectrometer (ICP-AES) to measure the release of metal ions, such as nickel. The results of ICP-AES analysis showed that TiAlN coated on a Ni-based alloy effectively reduced metal ion release levels, as compared to the uncoated Ni-based alloy. The effect of the nitride films was also reflected in corrosion resistance and the corrosion rate. It was demonstrated that the TiAlN-coated Ni-based alloy revealed a higher corrosion resistance than uncoated substrates. Furthermore, the impedance of nitride films on a Ni-based alloy was larger than that of the metal’s charge transfer reaction at short-term immersion.     

Native protein proteolysis in an immobilized enzyme reactor as a function of temperature

Trypsin concentration and the unmasking of cleavage sites in proteins play important roles in the stoichiometry of peptide production and the number of limit peptides generated during proteolysis. The hypothesis explored in this work was that native proteins could be digested and identified without disulfide reduction by (i) enhancing the unmasking of cleavage sites through elevated reaction temperatures and (ii) increasing trypsin concentration by use of an immobilized enzyme reactor (IMER). Transferrin was chosen as a model protein for these studies on the basis of its resistance to trypsin digestion. Results from this study showed greater than 70% sequence coverage in the peptides identified when nonreduced transferrin was digested at 60 °C. Large numbers of missed cleavages were observed from specific regions in proteins. Proteolysis appeared to start at a small number of high frequency cleavage sites in the cases of both reduced and nonreduced transferrin. Although approximately the same number of peptides were obtained from both structural forms of transferrin, the location of high frequency cleavage sites and the peptides produced were very different. Results from this study suggest that the location of initial cleavage sites along with the path of subsequent digestion depends strongly on the type of treatment used to open protein structures up for proteolysis.     

Compact ellipsometer employing a static polarimeter module with arrayed polarizer and wave-plate elements

A portable ellipsometer with a compact static polarimeter using an arrayed polarizer, an arrayed wave plate, and a CCD image sensor is developed. A high level of repeatability at a measurement speed of 0.3 s is demonstrated by measurement of SiO(2) films ranging from 2 to 300 nm in thickness deposited on an Si wafer. There is the potential to realize an ultracompact ellipsometer module by integrating the optical source and receiver, suitable for deployment in a variety of manufacturing equipment and measurement instruments.     

Strength improvement in unidirectional arrayed chopped strands with interlaminar toughening

The purpose of this study is to improve the strength of unidirectionally arrayed chopped strands (UACS), a sheet-like molding material made of regularly and unidirectionally arrayed discontinuous fiber strands impregnated with unsaturated thermoset resin. In a previous paper, we found that the final failure of UACS laminates is mainly controlled by the onset of unstable delamination growth. This paper presents interlaminar toughening of UACS laminates as a means of suppressing delamination. Specifically, we applied the following three approaches to toughening: (1) toughening the matrix resin itself, (2) inserting a toughened interlaminar layer, and (3) localizing the toughened layer only around the ends of the chopped strands. As a result, we demonstrate a tensile strength improvement of up to about 20%.     

3D protein microarrays: performing multiplex immunoassays on a single chip

The enzyme-linked immunosorbent assay (ELISA) is typically applied in the format of microtiter plates. To increase throughput and reduce consumption of precious samples, efforts have been made to transfer ELISA to the microchip format using conventional microarrays, microfluidic systems, and chips bearing microwells. However, all three formats lack the possibility to screen several analytes on several immobilized binders at a time or require complicated liquid handling, surface modifications, and additional equipment. Here, we describe an immunoassay performed on a standard microscope slide without the requirement for wells or tubes to separate the samples using standard surfaces and machinery already available for microarray technology. The new multiple spotting technique (MIST) comprises immobilization of a binder onto a surface and subsequent spotting of the second compound on the same spot, on top of the immobilized binder. We show that the analytes bind their ligands immediately within the confined space of separate droplets on the chip surface, thereby eliminating the need for extra incubation time. We illustrate the feasibility of the new technique by spotting dilution rows of proteins or monoclonal and polyclonal antibodies on top of their immobilized binders. Moreover, we demonstrate specificity by applying a mixture of antibodies in a multiplex format and demonstrate that the technique is compatible with conventional microarray protocols, such as total incubation. Finally, we indicate that the technique is capable of quantifying as little as 400 zmol (240,000 molecules) of analyte.