Rapid immunoassay exploiting nanoparticles and micromagnets: proof-of-concept using ovalbumin model.

AIM: We present a fast magnetic immunoassay, combining magnetic nanoparticles and micromagnets. High magnetic field gradients from micromagnets are used to develop a new approach to the standard ELISA. Materials & methods/results: A proof-of-concept based on colorimetric quantification of antiovalbumin antibody in buffer is performed and compared with an ELISA. After optimization, the magnetic immunoassay exhibits a limit of detection (40 ng/ml) and a dynamic range (40-2500 ng/ml) similar to that of ELISAs developed using same biochemical tools. CONCLUSION: Micromagnets can be fully integrated in multiwell plates at low cost to allow the efficient capture of immunocomplexes carried by magnetic nanoparticles. The method is generic and permits to perform magnetic ELISA in 30 min.

Surface chemistry at the nanometer scale influences insulin aggregation.

We synthesized surfaces with different hydrophobicities and roughness by forming self-assembled monolayers (SAMs) of mixed amine and octyl silanes. Insulin aggregation kinetics in the presence of the above surfaces is characterized by a typical lag phase and growth rate. We show that the lag time but not the growth rate varies as a function of the amine fraction on the surface. The amount of adsorbed protein and the adsorption rate during the aggregation process also vary with the amine fraction on the surface and are maximal for equal parts of amine and octyl groups. For all surfaces, the growth phase starts for identical amounts of adsorbed insulin. The initial surface roughness determines the rate at which protein adsorption occurs and hence the time to accumulate enough protein to form aggregation nuclei. In addition, the surface chemistry and topography influence the morphology of aggregates adsorbed on the material surface and the secondary structures of final aggregates released in solution.

Synchronization of Dictyostelium discoideum adhesion and spreading using electrostatic forces.

Synchronization of cell spreading is valuable for the study of molecular events involved in the formation of adhesive contacts with the substrate. At a low ionic concentration (0.17 mM) Dictyostelium discoideum cells levitate over negatively charged surfaces due to electrostatic repulsion. First, a two-chamber device, divided by a porous membrane, allows to quickly increase the ionic concentration around the levitating cells. In this way, a good synchronization was obtained, the onsets of cell spreading being separated by less than 5 s. Secondly applying a high potential pulse (2.5 V/Ref, 0.1s) to an Indium Tin Oxide surface attracts the cells toward the surface where they synchronously spread as monitored by LimE(Deltacoil)-GFP. During spreading, actin polymerizes in series of active spots. On average, the first spot appears 8-11s after the electric pulse and the next ones appear regularly, separated by about 10s. Synchronized actin-polymerization activity continues for 40s. Using an electric pulse to control the exact time point at which cells contact the surface has allowed for the first time to quantify the cellular response time for actin polymerization. Electrochemical synchronization is therefore a valuable tool to study intracellular responses to contact.

Peptide immobilization on amine-terminated boron-doped diamond surfaces.

This paper reports on the formation and characterization of semicarbazide termination on aminated boron-doped diamond (BDD) surfaces, and further preparation of peptide microarray through site-specific alpha-oxo semicarbazone ligation. Hydrogen-terminated BDD electrodes were first aminated using NH3 plasma treatment and then reacted with triphosgene and Fmoc-protected hydrazine to yield a protected semicarbazide termination. Subsequent deprotection and chemical reaction with glyoxylyl peptides led to the covalent immobilization of the peptides on the surface through site-specific ligation. The resulting surfaces were characterized using X-ray photoelectron spectroscopy (XPS) and fluorescence measurements.

Raman imaging and Kelvin probe microscopy for the examination of the heterogeneity of doping in polycrystalline boron-doped diamond electrodes.

The issue of the heterogeneity of boron doping in microcrystalline diamond films was addressed by four different methods: micro-Raman spectroscopy and Raman imaging, Kelvin probe force microscopy, conducting atomic force microscopy, and scanning electrochemical microscopy. The samples were commercially available films from Windsor Scientific, with an average boron concentration of about 5 x 10(20) cm(-3). In agreement with previous works, all of the methods showed that the boron uptake was nonuniform across the surface of the electrode. Two different types of regions were evidenced, with metallic or semiconducting properties that were characterized with different types of Raman spectra. The line shape of these spectra was strongly dependent on the excitation wavelength. Local variations in electroactivity were evidenced by the SECM curves, which are related to the electronic properties of the individual grains, which, in turn, are governed by the boron content of the individual crystallites. In this study, two different micro-Raman imaging techniques were used that reveal the grain structure of the films: the images constructed from the diamond line intensity perfectly reproduced the optical image obtained by illuminating the sample in reflection. The method also allows detection of the presence of nondiamond carbon, especially in the metallic parts of the samples. Other spectral features (intensity of the boron-related broad lines, as well as the frequency and width of the diamond line) were used to construct images. In every case, the grain structure of the film was revealed, as well as twinning within individual crystallites. All approaches revealed that no enhanced doping or boron depletion occurred at the grain boundaries.

Biotin grafting on boron-doped diamond.

Grafting of biotin on top of a polycrystalline boron-doped diamond layer was achieved by surface oxidation followed by an esterification reaction and revealed by fluorescently labelled streptavidin.