Ultrasensitive immuno-detection using viral nanoparticles with modular assembly using genetically-directed biotinylation

We report a novel, modular approach to immuno-detection based on antibody recognition and PCR read-out that employs antibody-conjugated bacteriophage, easily-manipulated nonpathogenic viruses, as affinity agents. Our platform employs phage genetically tagged for in vivo biotinylation during phage maturation that can easily be linked, through avidin, to any biotinylatable affinity agent, including full-length antibodies, peptides, lectins or aptamers. The presence of analyte is reported with high sensitivity through real-time PCR. This approach avoids the need to clone antibody-encoding DNA fragments, allows the use of full-length, high affinity antibodies and, by having DNA reporters naturally encapsulated inside the bacteriophage, greatly reduces nonspecific binding of DNA. We validate the efficacy of this new approach through the detection of VEGF (Vascular Endothelial Growth Factor), a known angiogenic cancer biomarker protein, at attomolar concentrations in bronchoalveolar lavage (BAL) fluid

High-Throughput Top-Down Fabrication of Uniform Magnetic Particles

Ion Beam Aperture Array Lithography was applied to top-down fabrication of large dense (108–109 particles/cm2) arrays of uniform micron-scale particles at rates hundreds of times faster than electron beam lithography. In this process, a large array of helium ion beamlets is formed when a stencil mask containing an array of circular openings is illuminated by a broad beam of energetic (5–8 keV) ions, and is used to write arrays of specific repetitive patterns. A commercial 5-micrometer metal mesh was used as a stencil mask; the mesh size was adjusted by shrinking the stencil openings using conformal sputter-deposition of copper. Thermal evaporation from multiple sources was utilized to form magnetic particles of varied size and thickness, including alternating layers of gold and permalloy. Evaporation of permalloy layers in the presence of a magnetic field allowed creation of particles with uniform magnetic properties and pre-determined magnetization direction. The magnetic properties of the resulting particles were characterized by Vibrating Sample Magnetometry. Since the orientation of the particles on the substrate before release into suspension is known, the orientation-dependent magnetic properties of the particles could be determined

Phenological shifts of abiotic events, producers and consumers across a continent

Ongoing climate change can shift organism phenology in ways that vary depending on species, habitats and climate factors studied. To probe for large-scale patterns in associated phenological change, we use 70,709 observations from six decades of systematic monitoring across the former Union of Soviet Socialist Republics. Among 110 phenological events related to plants, birds, insects, amphibians and fungi, we find a mosaic of change, defying simple predictions of earlier springs, later autumns and stronger changes at higher latitudes and elevations. Site mean temperature emerged as a strong predictor of local phenology, but the magnitude and direction of change varied with trophic level and the relative timing of an event. Beyond temperature-associated variation, we uncover high variation among both sites and years, with some sites being characterized by disproportionately long seasons and others by short ones. Our findings emphasize concerns regarding ecosystem integrity and highlight the difficulty of predicting climate change outcomes. The authors use systematic monitoring across the former USSR to investigate phenological changes across taxa. The long-term mean temperature of a site emerged as a strong predictor of phenological change, with further imprints of trophic level, event timing, site, year and biotic interactions.Peer reviewe

Chronicles of nature calendar, a long-term and large-scale multitaxon database on phenology

We present an extensive, large-scale, long-term and multitaxon database on phenological and climatic variation, involving 506,186 observation dates acquired in 471 localities in Russian Federation, Ukraine, Uzbekistan, Belarus and Kyrgyzstan. The data cover the period 1890-2018, with 96% of the data being from 1960 onwards. The database is rich in plants, birds and climatic events, but also includes insects, amphibians, reptiles and fungi. The database includes multiple events per species, such as the onset days of leaf unfolding and leaf fall for plants, and the days for first spring and last autumn occurrences for birds. The data were acquired using standardized methods by permanent staff of national parks and nature reserves (87% of the data) and members of a phenological observation network (13% of the data). The database is valuable for exploring how species respond in their phenology to climate change. Large-scale analyses of spatial variation in phenological response can help to better predict the consequences of species and community responses to climate change.Peer reviewe

High-sensitivity Protein Detection Using Immuno-PCR Phage Construct

Advances in the identification of novel biomarkers of cancer and infection are creating an increasing need for detection systems with superior sensitivity. Early detection and identification of infections speeds effective treatment, and cancers for which early diagnostic methods are available have distinctly higher 5-year survival rates than those for which early detection is lacking. Enzyme-linked immunosorbent assays (ELISAs) provide a high level of versatility, but often lack sensitivity for targets in the sub-picomolar concentration range. The current work describes the development of an ultrasensitive immuno-PCR protein detection assay. Bacteriophage biotin-coupled to off-the-shelf antibodies and quantified by real-time PCR are used as the reporter in the assay to detect the Vascular Endothelial Growth Factor (VEGF) in solution. VEGF promotes angiogenesis, is responsible for cell migration, inhibits cell death, and appears at elevated levels in cancer. The general experimental set-up can be modified based on top-down and bottom-up approaches. The top-down approach involves capturing VEGF on particles functionalized with anti-VEGF antibodies, recognition of VEGF by a monoclonal biotinylated antibody, attachment of NeutrAvidin, and the attachment of biotinylated phage onto the NeutrAvidin. The bottom-up approach involves binding a pre-made conjugate of biotinylated phage/NeutrAvidin/biotinylated antibody to VEGF captured on magnetic beads in a one-step reaction. The experiments are performed in phosphate buffer saline (PBS), 20% serum, and 50% bronchoalveolar lavage (BAL). VEGF can be reproducibly detected at concentrations down to 26 fM in PBS, 50% BAL, and 20% serum. In the second part of this work, VEGF is assayed on the sample-contact component of biosensors, amorphous pinhole-free aluminum oxide (alumina) coated surfaces, which represents an alternative way to detect phage reporters. This protective layer is usually used for insulation purposes in biosensors to protect them from corrosion in liquid, salty environments. Immobilization of biological agents on biosensors is a common practice and was achieved using TESBA–based silane chemistry coupled with protein A/G to properly orient the antibodies on the surface. VEGF is assayed at nano- and picomolar concentrations and the detection limit by ELISA is estimated to be 7.2 pM.Biomedical Engineering, Department o

One-Pot Synthesis of N-(Phosphorylmethyl)Pyrrolidines via Acid-Catalyzed Cascade Elimination/Cyclization/Friedel–Crafts Reaction

International audienceThe efficient approach to novel water-soluble 2-aryl-substituted N-(phosphorylmethyl)pyrrolidines via acid-catalyzed elimination/cyclization/Friedel–Crafts reaction sequence of {[(4,4-diethoxybutyl)amino]methyl}phosphonates is proposed. Zwitterionic structure of N-(phosphorylmethyl) pyrrolidines has been proved by X-ray crystallography

SEM image of 1micron (A), 300 nm (B), rod-shaped particles (C) and their corresponding VSM measurements (D, E, and F).

<p>The shape anisotropy dominates the material properties of the 300 nm thick evaporated particles, resulting in higher coercivity and lower remnant magnetization. The results of VSM measurements of rod-shaped particles show the highest coercivity among all particles, measuring as high as 37 Oe and remnant magnetization decrease from 0.7 to 0.3 with increase of layer thickness.</p

Summary of coercivity and remnant magnetization of magnetic particles with permalloy layers 40–300 nm thick.

<p>Summary of coercivity and remnant magnetization of magnetic particles with permalloy layers 40–300 nm thick.</p

Coercivity curves for 5 µm, 3 µm, 1 µm, 300 nm, and rod-shaped particles.

<p>The coercivity (A) and the remnant magnetization (B) values were extracted from the VSM plots described above for different particle sizes and varying magnetic layer thicknesses.</p

Particle Fabrication Sequence.

<p>Spin-coating of PMGI and PMMA on a clean silicon wafer (a) is followed by exposure of the sample to a broad beam of helium ions through a stencil mask to form the pattern (b). During development, the exposed areas of PMMA wash away, and a subsequent etch in TMAH removes the PMGI layer underneath the PMMA openings (c). The particles of interest are evaporated as stacked layers of 10 nm gold, 10 nm of permalloy, and 10 nm gold (d). A lift-off procedure removes the evaporated metal on top of the PMMA layer (e) and the PMGI layer is etched in TMAH solution (f) to release the particles.</p