DDI-microFIA--A readily configurable microarray-fluorescence immunoassay based on DNA-directed immobilization of proteins

We describe a chip-based immunoassay for multiplex antigen detection, based on the self-assembly of semi-synthetic DNA-protein conjugates to generate an easily configurable protein microarray. The general principle of this microarray-fluorescence immunoassay (microFIA) is similar to that of a two-sided (sandwich) immunoassay. However, covalent single-stranded DNA-streptavidin conjugates are employed for the efficient immobilization of biotinylated capture antibodies through hybridization to complementary surface-bound DNA oligomers. In a model system, we use the DNA-directed immobilization (DDI) of antibodies to generate an antibody microarray for the parallel detection of the tumor marker human carcinoembryonic antigen (CEA), recombinant mistletoe lectin rViscumin (rVis), ceruloplasmin (CEP), and complement-1-inactivator (C1A) in human blood serum samples. Detection limits down to 400 pg mL(-1) are reached. In addition, we describe a method for the internal standardization of protein microarray analyses, based on the simultaneous measurement of constant amounts of the blood proteins CEP and C1 A, intrinsically present in human serum, to compensate for interexperimental variations usually occurring in microarray analyses. The standardization leads to a significantly higher data reliability and reproducibility in intra- and interassay measurements. We further demonstrate that the DDI-microFIA can also be carried out in a single step by tagging of the analyte simultaneously with both capture and detection antibody and subsequent immobilization of the immunocomplex formed, on the DNA microarray capture matrix. This protocol significantly reduces handling time and costs of analysis.     

Synthesis of new boron-rich building blocks for boron neutron capture therapy or energy-filtering transmission electron microscopy

The synthesis of a new ortho-carborane derivative, tetracarboranylketone 4, is reported here. Ketone 4 was prepared from a tetraalkynylated ketone by the addition of decaborane. The keto group was then easily modified to yield the glycosides 17alpha and 18beta, which contain glucose or galactose, respectively, and the nucleotide 13b. In addition to ketone 4, which is acyclic, cyclic ketone 8 was also synthesised. X-ray diffraction analysis of compound 4 indicated the presence of two toluene guest molecules per molecule of the host compound. Furthermore, compound 4 displays a rather low cytotoxicity. These novel products can be used as building blocks to create a new class of biomolecules containing high-density carborane clusters. Such molecules may constitute powerful tools for applications like Boron Neutron Capture Therapy or Energy-Filtering Transmission Electron Microscopy.     

Different Enzymatic Processing of γ‐Phosphoramidate and γ‐Phosphoester‐Modified ATP Analogues

Monitoring the activity of ATP‐consuming enzymes provides the basis for elucidating their modes of action and regulation. Although a number of ATP analogues have been developed for this, their scope is restricted because of the limited acceptance by respective enzymes. In order to clarify which kind of phosphate‐modified ATP analogues are accepted by the α‐β‐phosphoanhydride‐cleaving ubiquitin‐activating enzyme 1 (UBA1) and the β‐γ‐phosphoanhydride‐cleaving focal adhesion kinase (FAK), we tested phosphoramidate‐ and phosphoester‐modified ATP analogues. UBA1 and FAK were able to convert phosphoramidate‐modified ATP analogues, even with a bulky modification like biotin. In contrast, a phosphoester‐modified analogue was poorly accepted. These results demonstrate that minor variations in the design of ATP analogues for monitoring ATP utilization have a significant impact on enzymatic acceptance.     

In-service performance assessment of electrostatic precipitators serving a rubber vulcanization process

We examined two emission abatement systems of some vulcanization ovens, serving a unit producing small rubber-based parts for automotive application. Each emission control unit treats the gases exhausted by three to five ovens. A heat exchanger cools down the fumes to a temperature suitable for the correct operation of a couple of two-stage electrostatic precipitators in series. We performed quantitative analysis of concentrations and size distributions in these rubber fumes using aerosol technology instrumentation, namely optical particle spectrometers and electrical mobility particle sizers. The size of sampled particles was mainly between 100?nm and 1000?nm. We evaluated the performance of the exhaust fume abatement units, with focus on the electrostatic precipitator. Concerning batch ovens, the quantitative trend of the emissions follows the thermal cycle of the post-curing process. Time interval since the last maintenance operation causes a gradual reduction in the removal efficiency. The measured data demonstrate the reliability and the adequacy of aerosol instrumentation for the characterization of the emissions from rubber vulcanization ovens. The pair of electrostatic precipitators was shown to be effective in removing most of the particles detected in the fumes stream. The measurement protocol developed in this study allows assessing the influence of the maintenance schedule on the performance of the emission control units. New technologies for treating organic vapors can be evaluated in a reliable and effective way.

Copyright © 2019 American Association for Aerosol Research     

Understanding selective oxidations

Functionalizing organic molecules is an important value-creating step throughout the entire chemical value-chain. Oxyfunctionalization of e.g. C-H or C=C bonds is one of the most important functionalization technologies used industrially. The major challenge in this field is the prevention of side reactions and/or the consecutive over-oxidation of the desired products. Despite its importance, a fundamental understanding of the intrinsic chemistry, and the subsequent design of a tailored engineering environment, is often missing. Industrial oxidation processes are indeed to a large extent based on empirical know-how. In this mini-review, we summarize some of our previous work to help to bridge this knowledge gap and elaborate on our ongoing research.     

Screening for Cytochrome P450 Reactivity by Harnessing Catalase as Reporter Enzyme

The big screen : We have devised a high‐throughput screening method for organic peroxide‐dependent P450 reactivity by taking advantage of a previously undescribed activity of catalase, which was used as reporter enzyme. This two‐step assay, followed by liquid chromatography/mass spectrometry analyses, allowed the facile identification of several new substrates for bacterial P450 enzymes.

     

A Device for Zero-Eye-Movement Reading

A portable personal computer has been programmed as a flexible and economical text display device capable of delivering single and multiple words or symbols flashed in the center of the screen with a wide range of repetition times and duty cycles (fraction of stimulus on/off time). All aspects of a computer-controlled experiment are controlled by a command file which can be created by workers with basic word processing skills and no knowledge of computer programming. Digital output is provided for recording stimulus timing on a chart recorder or computer for comparison to eye movements or other externally recorded variables. The system is ideal for zero-eye-movement reading experiments in which sentences are flashed a word or group of words at a time in the center of a CRT screen. If the subject is then asked to repeat the sentence aloud, the percentage of words correctly recalled is a measure of "reading comprehension." Results with nine subjects match previous data [1] obtained cinematographically.     

High-speed cinematographic recording and numerical simulation of particles depositing on electret fibres

A method is described for visualization and recording, as well as for numerical simulation of particle motion during the deposition onto single electrically charged filter fibres (electret fibres). For this a microscope and a high-speed cine camera are used to record the trajectories of particles in the process of flowing around, and being deposited on, a fibre (two-dimensionally in a plane perpendicular to the fibre axis). The particle trajectories are then modeled, based on the experimental data and suitable mathematical models for the fluid flow and the electric fields of the fibre charges. As the fibre charge density, in general, is not known, it is used as a fitting parameter in the model calculation. Using the example of split-fibres with a rectangular cross-section and a bipolar electric charge it is shown that, using models known from the literature, very good agreement can be achieved between experiment and simulation. With this electret fibre type, the trajectory simulations, assuming an ideal bipolar charge configuration, gave effective charge densities between roughly 100 and 500 μAs m⁻², corresponding to total surface charge densities between 230 and 1150 μAs m⁻².     

Computing optical flow across multiple scales: An adaptive coarse-to-fine strategy

Single-scale approaches to the determination of the optical flow field from the time-varying brightness pattern assume that spatio-temporal discretization is adequate for representing the patterns and motions in a scene. However, the choice of an appropriate spatial resolution is subject to conflicting, scene-dependent, constraints. In intensity-base methods for recovering optical flow, derivative estimation is more accurate for long wavelengths and slow velocities (with respect to the spatial and temporal discretization steps). On the contrary, short wavelengths and fast motions are required in order to reduce the errors caused by noise in the image acquisition and quantization process.Estimating motion across different spatial scales should ameliorate this problem. However, homogeneous multiscale approaches, such as the standard multigrid algorithm, do not improve this situation, because an optimal velocity estimate at a given spatial scale is likely to be corrupted at a finer scale. We propose an adaptive multiscale method, where the discretization scale is chosen locally according to an estimate of the relative error in the velocity estimation, based on image properties.Results for synthetic and video-acquired images show that our coarse-to-fine method, fully parallel at each scale, provides substantially better estimates of optical flow than do conventional algorithms, while adding little computational cost.