Antibody-based surfaces: rapid characterization using two complementary colorimetric assays

Finding a general solution for optimizing the grafting of antibody on solid surfaces is difficult due to the variety of material, grafting principles and chemistries or surface formats available (beads, microplates, fibers, etc.). Pre-screening methods able to assess grafting efficiency (GE) and specific activity (SA) are required. In this context, we present here two colorimetric assays that can be used on a wide variety of surface format, chemistry, etc. The first one, ADECA (Amino Density Estimation by Colorimetric Assay) allows a rapid estimation of grafted antibodies and allows calculating the GE. The second one, A₂HRP (Antibody Anti-HorseRadish Peroxidase) provides a measure of the amount of active antibody, which, combined to ADECA, is used to determine the SA of grafted antibody. Analytical parameters (limit of detection, repeatability, linearity, etc.) of these two colorimetric assays are presented. Using two commercially available microplates, we demonstrated that, when used in parallel, these rapid and sensitive methods are well adapted to pre-screening of antibody grafting performances.     

Evaluation of pulsed radiofrequency glow discharge time-of-flight mass spectrometry for precious metal determination in lead fire assay buttons

In this paper, an exploration of the capabilities and limitations of pulsed radiofrequency glow discharge time-of-flight mass spectrometry (GD-TOFMS) for the determination of the precious metals Ag, Au, Pd, Pt and Rh in lead buttons obtained by Pb fire assay is reported on. Since the matrix consists almost entirely of lead (>99%), the occurrence of doubly charged Pb (Pb²⁺) ions can hinder accurate determination of Rh. This problem was counteracted by relying on the time-resolved formation of different ion types over the pulse period of the glow discharge, which allows discrimination against the Pb²⁺ ions. The formation of ArCu⁺ ions as a result of the use of a copper anode was assessed to pose no threat to the accuracy of the results obtained for the set of samples analyzed as its contribution to the total signal at m/z = 103 could be adequately corrected for. The method developed was evaluated in terms of accuracy and precision using a set of Pb button standards with analyte concentrations between 5 and 100 μg g⁻¹. For the purpose of validation, a 10 μg g⁻¹ standard was considered as a sample. Overall, an acceptable accuracy was obtained with a bias of <5% between the experimental results and the corresponding reference values, except for Au, for which a larger deviation occurred. Precision values (repeatability) of typically <5% relative standard deviation (RSD) (for N = 3) were obtained and the limits of detection (LODs) vary from ∼0.020 μg g⁻¹ for Ag to ∼0.080 μg g⁻¹ for Pt.     

Inclined fire whirls

Air entrainment, leading to strong fire whirls, is commonly thought to be caused by the buoyant rise of the hot combustion products under the influence of gravity. We have, however, created in the laboratory steady, axisymmetric strong fire whirls with axes inclined 30° from the vertical orientation, whirls which model an inclined fire whirl, about 30 m tall, observed in California wildland near the Cleveland National Forest. The results contradict the common notion of buoyancy being significant for the structure of the whirl, implying that strong fire whirls instead are dominated by rotation only, even if their axis is vertical. The new concept of rotation-controlled fire whirls is explained by a Rossby number displacing the Froude number (or the Richardson number) in describing the phenomenon.     

Generating Encoded Compound Libraries for Fabricating Microarrays as a High-Throughput Protein Ligand Discovery Platform

We demonstrate an effective method for generating libraries of encoded compounds for fabricating large compound microarrays on solid supports. This method is based on one-bead, one-compound synthesis and employs a novel trilayer bead-partition scheme that ensures sufficient quantity of synthesized compounds releasable from each bead for compound microarray fabrication in high-throughput protein–ligand discovery assays.

[Supplementary materials are available for this article. Go to the publisher's online edition of Synthetic Communications® for the following free supplemental resource(s): Full experimental and spectral details.]     

Tunable fiber laser based photoacoustic gas sensor for early fire detection

A photoacoustic gas sensor using a near-infrared tunable fiber laser and based on wavelength modulation spectroscopy technique is developed. This sensor is capable of quasi-simultaneous quantification of water vapour, acetylene, carbon dioxide, and carbon monoxide (H₂O, C₂H₂, CO and CO₂) concentrations in the fire emulator. The feasibility of using this sensor as an early fire detector was demonstrated. The fire warning gases from smoldering paper were measured. The peak concentrations of gases from smoldering paper were 20,300 ppm H₂O, 2.1 ppm C₂H₂, 756 ppm CO, and 1612 ppm CO₂ after 400 s.     

REDUCTIVE REMOVAL OF HALOGEN BY THIOLATES FROM 1-HALOCYCLO-PROPYL SULFIDES

Abstract

Nucleophilic substitution of cyclopropyl halides or tosylates is often accompanied by ring rupture, but this can be prevented by the presence of an electron releasing substituent in α-position (NR₂, OR, SR). Alkylthio and arylthio groups are particularly effective in stabilizing intermediate cyclopropyl cations. These give with most nucleophiles e.g. alkoxide, azide, fluoride, in relatively rapid reactions good yields of substitution products.     

Effects of sequence context on the binding of tryptophan-containing peptides by the cucurbit[8]uril–methyl viologen complex

This paper describes a novel assay for measuring the relative extent of peptide binding in a large parallel format and the use of this assay to explore the effects of sequence context on the binding of tryptophan (Trp)-containing peptides by the synthetic receptor comprising the noncovalent complex between cucurbit[8]uril and methyl viologen (i.e. Q8√MV). The extent of quenching of Trp fluorescence upon binding to Q8√MV was used to measure the relative extent of binding and thus the relative affinities of 104 Trp-containing peptides, in parallel, using a fluorescence plate reader. This study resulted in the remarkable observation that the identity of the amino acid residues at positions adjacent to the Trp-binding site has little if any influence on the binding affinity. This finding suggests that Q8√MV should be effective for the recognition of Trp residues within a broad range of peptide sequences.     

Rapid detection of irradiated frozen hamburgers

DNA comet assay can be employed as a rapid and inexpensive screening test to check whether frozen ground beef patties (hamburgers) have been irradiated as a means to increase their safety by eliminating pathogenic bacteria, e.g. E. coli O157:H7. Such a detection procedure will provide an additional check on compliance with existing regulations, e.g. enforcement of labelling and rules in international trade. Frozen ready prepared hamburgers from the market place were ‘electron irradiated’ with doses of 0, 1.3, 2.7, 4.5 and 7.2 kGy covering the range of potential commercial irradiation. DNA fragmentation in the hamburgers was made visible within a few hours using the comet assay, and non-irradiated hamburgers could be easily discerned from the irradiated ones. Even after 9 months of frozen storage, irradiated hamburgers could be identified. Since DNA fragmentation may also occur with other food processes (e.g. temperature abuse), positive screening tests shall be confirmed using a validated method to specifically prove an irradiation treatment, e.g. EN 1784 or EN 1785.     

Detection of radiation treatment of beans using DNA comet assay

A simple technique of microgel electrophoresis of single cells (DNA Comet Assay) enabled a quick detection of radiation treatment of several kinds of leguminous beans (azuki, black, black eye, mung, pinto, red kidney and white beans). Each variety was exposed to radiation doses of 0.5, 1 and 5 kGy covering the permissible limits for insect disinfestation. The cells or nuclei from beans were extracted in cold PBS, embedded in agarose on microscope slides, lysed between 15 and 60 min in 2.5% SDS and electrophoresis was carried out at a voltage of 2 V/cm for 2–2.5 min. After silver staining, the slides were evaluated through an ordinary transmission microscope. In irradiated samples, fragmented DNA stretched towards the anode and the damaged cells appeared as a comet. The density of DNA in the tails increased with increasing radiation dose. However, in non-irradiated samples, the large molecules of DNA remained relatively intact and there was only minor or no migration of DNA; the cells were round or had very short tails only. Hence, the DNA comet assay provides an inexpensive, rapid and relatively simple screening method for the detection of irradiated beans.