A simple device for multiplex ELISA made from melt-extruded plastic microcapillary film

We present a simple device for multiplex quantitative enzyme-linked immunosorbant assays (ELISA) made from a novel melt-extruded microcapillary film (MCF) containing a parallel array of 200µm capillaries along its length. To make ELISA devices different protein antigens or antibodies were immobilised inside individual microcapillaries within long reels of MCF extruded from fluorinated ethylene propylene (FEP). Short pieces of coated film were cut and interfaced with a pipette, allowing sequential uptake of samples and detection solutions into all capillaries from a reagent well. As well as being simple to produce, these FEP MCF devices have excellent light transmittance allowing direct optical interrogation of the capillaries for simple signal quantification. Proof of concept experiments demonstrate both quantitative and multiplex assays in FEP MCF devices using a standard direct ELISA procedure and read using a flatbed scanner. This new multiplex immunoassay platform should find applications ranging from lab detection to point-of-care and field diagnostics.

The role of myeloid receptors on murine plasmacytoid dendritic cells in induction of type I interferon

This study tested the hypothesis that a set of predominantly myeloid restricted receptors (F4/80, CD36, Dectin-1, CD200 receptor and mannan binding lectins) and the broadly expressed CD200 played a role in a key function of plasmacytoid DC (pDC), virally induced type I interferon (IFN) production. The Dectin-1 ligands zymosan, glucan phosphate and the anti-Dectin-1 monoclonal antibody (mAb) 2A11 had no effect on influenza virus induced IFNα/β production by murine splenic pDC. However, mannan, a broad blocking reagent against mannose specific receptors, inhibited IFNα/β production by pDC in response to inactivated influenza virus. Moreover, viral glycoproteins (influenza virus haemagglutinin and HIV-1 gp120) stimulated IFNα/β production by splenocytes in a mannan-inhibitable manner, implicating the function of a lectin in glycoprotein induced IFN production. Lastly, the effect of CD200 on IFN induction was investigated. CD200 knock-out macrophages produced more IFNα than wild-type macrophages in response to polyI:C, a MyD88-independent stimulus, consistent with CD200's known inhibitory effect on myeloid cells. In contrast, blocking CD200 with an anti-CD200 mAb resulted in reduced IFNα production by pDC-containing splenocytes in response to CpG and influenza virus (MyD88-dependent stimuli). This suggests there could be a differential effect of CD200 on MyD88 dependent and independent IFN induction pathways in pDC and macrophages. This study supports the hypothesis that a mannan-inhibitable lectin and CD200 are involved in virally induced type I IFN induction.

A multi-level analytical approach for detection and visualization of intracellular NO production and nitrosation events using diaminofluoresceins

Diaminofluoresceins are widely used probes for detection and intracellular localization of NO formation in cultured/isolated cells and intact tissues. The fluorinated derivative, 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM), has gained increasing popularity in recent years due to its improved NO-sensitivity, pH-stability, and resistance to photo-bleaching compared to the first-generation compound, DAF-2. Detection of NO production by either reagent relies on conversion of the parent compound into a fluorescent triazole, DAF-FM-T and DAF-2-T, respectively. While this reaction is specific for NO and/or reactive nitrosating species, it is also affected by the presence of oxidants/antioxidants. Moreover, the reaction with other molecules can lead to the formation of fluorescent products other than the expected triazole. Thus additional controls and structural confirmation of the reaction products are essential. Using human red blood cells as an exemplary cellular system we here describe robust protocols for the analysis of intracellular DAF-FM-T formation using an array of fluorescence-based methods (laser-scanning fluorescence microscopy, flow cytometry and fluorimetry) and analytical separation techniques (reversed-phase HPLC and LC-MS/MS). When used in combination, these assays afford unequivocal identification of the fluorescent signal as being derived from NO and are applicable to most other cellular systems without or with only minor modifications.

Use of soft heterocyclic N‑donor ligands to separate actinides and lanthanides

The removal of the most long-lived radiotoxic elements from used nuclear fuel, minor actinides, is foreseen as an essential step toward increasing the public acceptance of nuclear energy as a key component of a low-carbon energy future. Once removed from the remaining used fuel, these elements can be used as fuel in their own right in fast reactors or converted into shorter-lived or stable elements by transmutation prior to geological disposal. The SANEX process is proposed to carry out this selective separation by solvent extraction. Recent efforts to develop reagents capable of separating the radioactive minor actinides from lanthanides as part of a future strategy for the management and reprocessing of used nuclear fuel are reviewed. The current strategies for the reprocessing of PUREX raffinate are summarized, and some guiding principles for the design of actinide-selective reagents are defined. The development and testing of different classes of solvent extraction reagent are then summarized, covering some of the earliest ligand designs right through to the current reagents of choice, bis(1,2,4-triazine) ligands. Finally, we summarize research aimed at developing a fundamental understanding of the underlying reasons for the excellent extraction capabilities and high actinide/lanthanide selectivities shown by this class of ligands and our recent efforts to immobilize these reagents onto solid phases.

N-Methylthiomethylation of benzimidazoles with DMSO and their chemoselective oxidation to sulfoxides with NaBiO3

A straightforward one-step method for the N-methylthiomethylation of benzimidazoles has been developed employing DMSO as a solvent and as a reagent. This methodology has been applied for the synthesis of diverse N-methylthiomethyl derivatives of benzimidazoles. The products can be chemoselectively oxidized to the corresponding sulfoxides with NaBiO3 in acetic acid. Both the N-methylthiomethyl derivatives of benzimidazoles and their corresponding sulfoxides are important medicinal scaffolds.

A bifurcated pathway to thiazoles and imidazoles using a modular flow microreactor

A scalable method for the preparation of 4,5-disubstituted thiazoles and imidazoles as distinct regioisomeric products using a modular flow microreactor has been devised. The process makes use of microfluidic reaction chips and packed immobilized-reagent columns to effect bifurcation of the reaction pathway.

Capillary assay device with internal hydrophilic coating

The present invention provides assay devices having a unitary body with an exterior surface, the unitary body being substantially transparent to visible light and formed from a material having a refractive index in the range 1.26 to 1.40, the refractive index being measured at 20 °C with light of wavelength 589 nm, and wherein the unitary body is formed from a hydrophobic material, and at least two capillary bores extending internally along the unitary body, wherein at least a portion of the surface of each capillary bore includes a hydrophilic layer for retaining an assay reagent, and wherein the hydrophilic layer is also substantially transparent to visible light to allow optical interrogation of the capillary bores through the capillary wall. The present invention also provides assay systems including such assay devices, methods of performing an assay using such assay devices and method of method for manufacturing such assay devices.